Oligothionucleotides

ABSTRACT

Chemical compounds consisting of an alpha or beta oligo-4&#39;-thioribonucleotide or oligo-4&#39;-thio-2&#39;-deoxyribonucleotide characterized in that they comprise a concatenation of 4&#39;-thioribonucleotides or 4&#39;-thio-2&#39;deoxyribonucleotides, respectively. The concatenation is optionally linked to an effector, in particular a radical corresponding to an intercalating agent or a photoactivatable or chemical radical, e.g. a radical carrying a function which reacts directly or indirectly with the nucleotide chains, or a radical whose presence enables easy and sensitive detection. Methods for preparing said compounds, and their uses in therapeutics, diagnostics and as laboratory reagents, are also described.

FIELD OF THE INVENTION

The present invention relates to new chemical compounds as well as theirapplications. The chemical compounds according to the present inventionare oligonucleotide compounds at least partially consisting of anoligo-4'-thioribonucleotide or an oligo-4'-thio-2'-deoxyribonucleotidecomprising a concatenation of 4'-thio-nucleotides.

BACKGROUND OF THE INVENTION

In French Patent Applications FR 8,301,223 (2,540,122) and FR 8,411,795(2,568,254) have been described chemical compounds consisting of anoligonucleotide or an oligodeoxynucleotide comprising a concatentationof natural or modified nucleotides, that is to say beta-nucleotides,onto which is attached by a covalent bond at least one intercalatinggroup, which possess the property of selectively blocking the expressionof a gene and which, because of this, are particularly useful in therapyas antiviral, antibiotic, antiparasitic or antitumor substances.

In International Application PCT WO 83/01451, has been described amethod for blocking the translation of messenger RNA (mRNA) into proteinby hybridization of the mRNA with an oligonucleotide having the sequencecomplementary to the mRNA, the oligonucleotide being stabilized inphosphotriester form.

In International Application WO 88/04301 have been describedoligonucleotides of alpha anomeric configuration having parallelpairings with complementary sequences.

Chemotherapy with antisense oligonucleotides relates to RNA or DNAtargets of all living organisms (cells, bacteria, parasites, viruses oroncogenes).

The use of synthetic antisense oligonucleotides having the samestructure as the natural nucleic acids is faced, mainly in biologicalmedium, with problems of sensitivity to nucleases and cell penetration.

To overcome these limitations, oligonucleotide analogs capable of beingmore resistant to nucleases and of penetrating better into the cellsacross the cyto-plasmic membrane have been synthesized.

There has indeed been described in the prior art derivatives ofoligonucleotide compounds resisting enzymatic degradations better, whosephosphate part was modified into thiophosphate or methyl phosphonateespecially. However, these derivatives exhibit a chirality at the levelof the phosphate capable of generating insoluble diastereoisomers.

SUMMARY OF THE INVENTION

The compounds according to the invention may have the natural anomericconfiguration beta or the non-natural anomeric configuration alpha.

Thus, alpha(a) and beta(b) 4'-thionucleotides are represented by theformulae(a) and (b) respectively ##STR1## (a) and (b) represent4'-thionucleosides, a phosphate should be added in 5' in order to obtainnucleotides.

Under these conditions, many uses for biological and evenpharmacological purposes already known for oligonucleotides can beenvisaged, and this with greater efficiency.

More precisely, the subject of the present invention is chemicalcompounds consisting of an oligo-4'-thioribonucleotide or anoligo-4'-thio-2'-deoxyribonucleotide, characterized in that theycomprise a concatenation of 4'-thioribonucleotides or4'-thio-2'-deoxyribonucleotides respectively, it being possible for saidconcatenation to be optionally linked to an effector, especially aradical corresponding to an intercalating agent or a chemical orphotoactivable radical, such as a radical carrying a functional groupwhich reacts directly or indirectly with the nucleotide chains or aradical whose presence permits easy and sensitive detection.

In particular, the subject of the invention is newoligo-4'-thionucleotide derivatives, their preparation and their useespecially as probes permitting the detection of a defined sequence ofnucleic acids, as artificial nucleases specific for DNA or RNA sequencesor alternatively as agents for selectively blocking the expression of agene, whether endogenous (for example oncogene gene) or exogenous (forexample DNA or RNA of viruses, parasites or bacteria).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the curve for thermal stability of the betadT₅ (S_(r)T)dT_(e) (SEQ ID NO: 3)/betadC₂ A₁₂ C₂ (SEQ ID NO: 4) duplex comparedwith that of the natural betadT₁₂ (SEQ ID NO: 3)/betadC₂ A₁₂ C₂ (SEQ IDNO: 4) duplex.

FIG. 2 represents the curve for the enzymatic digestion of beta(S_(r)T)dT₁₁ (SEQ ID NO: 3).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a new class of chimeric oligonucleotides,the 4'-thiooligonucleotides, in which the intracyclic oxygen of thefuranose ring has been replaced by a sulfur atom.

It has indeed now been found, and this is what constitutes the subjectof the present invention, that the substitution of oxygen by a sulfur onthe sugar part of the nucleosides rather than on the phosphate group, onthe one hand, ensures the solubility of the oligomers and, on the otherhand, could increase their penetration into the target cell, thesubstitution of oxygen by the sulfur element rendering the molecule morelipophilic.

The derivatives of 4'-thionucleotides form hybridization complexes withthe sequences complementary to RNA which are much more stable than thoseformed with DNA and that, with respect to RNAs, the derivatives of4'-thionucleotides form hybridization complexes which are more stablethan the derivatives of natural beta-nucleotides.

Among the compounds, according to the present invention, there may bementioned more particularly the beta oligomeric compounds of formula##STR2## in which

the B radicals may be identical or different and each represent a baseof a nucleic acid optionally modified, activable and/or comprising anintercalating group;

the X radicals may be identical or different and each represent anoxoanion O⁻, a thioanion S⁻, an alkyl group, an alkoxy or aryloxy group,an aminoalkyl group, an aminoalkoxy group, a thioalkyl group, an alkylor alkoxy radical substituted by a nitrogen-containing heterocycle or a--Y--Z group;

R and R', which may be identical or different, each represent a hydrogenatom or a --Y--Z or Y'--Z' group;

Y and Y', which are identical or different, each represent a straight orbranched alkylene radical --alk-- or a radical chosen from ##STR3##

In particular, Y and Y' represent a radical chosen from ##STR4## withU=O, S or N

E may have the same meanings as X except Y--Z or Y'--Z';

L represents O, S or --NH--;

n and n' represent an integer including 0;

J represents a hydrogen atom or an hydroxyl group;

Z and Z', which are identical or different, each represent OH or aradical corresponding to an effector, especially a radical correspondingto an intercalating agent or a radical carrying a functional group whichreacts directly or indirectly with the nucleotide chains or a radicalwhose presence permits easy and sensitive detection.

In this formula I, the following condensed representation of nucleotidesis used: ##STR5## which corresponds to the structural formula: ##STR6##in which the (3') and (5') ends have been stated.

It should be noted that the formula I represents a concatenation of4'-thionucleotides which may be identical or different, of D or Lconfiguration, n simply indicating the number of nucleotides included inthe molecule; n is preferably a number between 1 and 50, and still morepreferably between 1 and 25.

There may be mentioned in particular the products for which L representsoxygen; R and R' represent hydrogen and B a natural nucleic base, eitheradenine, thymine, cytosine or guanine when J=H and adenine, uracil,cytosine and guanine when J=OH.

The intercalating agents are products known in the techniques relatingto nucleic acids; they are compounds capable of "becoming intercalated"in the structure of DNAs or RNAs, that is to say capable of becominginserted between the base plates of nucleic acids.

The intercalating agents may be chosen from the polycyclic compoundshaving a planar configuration such as acridine and its derivatives,furocoumarin and its derivatives, daunomycin and the other derivativesof anthracycline, 1,10-phenanthroline, phenanthridine and itsderivatives, proflavine, porphyrins, derivatives ofdipyrido[1,2-a:3',2'-d]imidazole, ellipticine or ellipticinium and theirderivatives and diazapyrene and its derivatives.

The reactive chemical radicals may be radicals which can react directlyor indirectly with a nucleotide chain to form a covalent bond or inorder to modify it chemically, or in order to cut it. Preferably, thesereactive chemical radicals are activable, for example, chemically,biochemically or photochemically.

The activable reactive radicals may be chosen fromethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid,porphyrins, 1,10-phenanthroline, 4-azidoacetophenone, ethylene-imine,beta-chloro-ethylamine, psoralen and their derivatives, and the aromaticcompounds absorbing near-ultraviolet or visible radiations or which canreact chemically with the nucleic constituents.

More particularly, the radicals which are chemically activable in thepresence of metal ions, oxygen and a reducing agent(ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid,porphyrin, phenanthroline) induce the cutting in nucleic acid sequencessituated in their vicinity.

By irradiation in the visible or near-ultraviolet region, it is possibleto activate the derivatives which absorb these radiations and to carryout bridging reactions or photoinduced reactions (cutting andmodification of the nucleic bases) of the nucleic acids on which isattached the oligothionucleotide carrying the activable group.

Among the Z and Z' radicals, there may be mentioned more particularly:

the radicals derived from ethylenediaminetetraacetic acid of formula:##STR7## the radicals derived from diethylenetriaminepentaacetic acid,the radicals derived from methylpyrroporphyrin of formula: ##STR8## theradicals derived from phenanthroline of formula: ##STR9## the radicalsderived from acridine: ##STR10## the radicals derived from proflavine##STR11##

R representing an amino (NH₂) or azido (N₃) group

the radicals derived from biotin ##STR12## the radicals derived from4-azidoacetophenone of formula: ##STR13##

The B radical may be preferably chosen from the natural nucleic bases(thymine, adenins, cytosine, guanine, uracil) but it is possible to usemodified nucleic bases. There may be mentioned 2-amino adenine and itsderivatives substituted for example on the nitrogen atom N⁶ by anaminoalkylene radical or by an azidophenylalkylene radical, guaninesubstituted on the oxygen atom O⁶ for example by a(w-alkylene)-9-acridine group, (w-aminoalkyl)amino-8-adenine and itsderivatives substituted on the amino radical in w by an acridine group,or the halogenated or azide-containing derivatives such as 5-bromouracil, 8-azidoadenine, 7-deazaadenine and 7-deazaguanine. It is alsopossible to use derivatives of nucleic bases comprising an intercalatinggroup or a chemically or photochemically activable group.

Thus, the functionalization of C or T by an aziridine group in position4 leads to the formation of CH₂ -CH₂ covalent bridges between the twocomplementary strands on G and A respectively. Therefore, moreparticularly, the B radical is then chosen from 4-azidocytosine or4-azidothymine.

Preferably, the X radical represents an oxoanion. However, the X radicalmay represent an alkyl radical containing 1 to 7 carbon atoms (methyl,ethyl, propyl), an alkoxy radical whose alkyl part contains 1 to 7carbon atoms (methoxy, ethoxy, 2,2-dimethylpropyloxy), an aminoalkyl oraminoalkoxy radical of general formula R₁ R₂ N--alk--A-- in which Arepresents a bond or an oxygen atom, --alk-- represents an alkyleneradical containing 1 to 10 carbon atoms and R₁ and R₂, which areidentical or different, represent a hydrogen atom or an alkyl radicalcontaining 1 to 7 carbon atoms or form, together with the nitrogen atomto which they are bonded, a 5- or 6-membered saturatednitrogen-containing heterocycle, it being understood that the R₁ R₂N-group may be quaternized, or an alkylthio radical whose alkyl partcontains 1 to 7 carbon atoms.

In the formula (I), the --alk-- radical is preferably a straight orbranched alkylene radical having 1 to 10 carbon atoms.

In particular, from the 3' or 5' terminal alcohol functional groups ofthe oligothionucleotide, the effector Z can therefore by introduced viaa chain (CH₂)_(n) linked to a functionalization Z', of diverse natures,to the glycoside part of the oligomer.

From the following formula: ##STR14## there will be obtained, dependingon what Z'₁ represents, for example

a phosphate or methyl phosphonate of formula ##STR15## U=O, N or S

an ether of general formula ##STR16##

an ester of general formula: ##STR17## or alternatively

a carbamate of general formula: ##STR18## In general, n' is between 1and 10.

The present invention also relates to the preceding compounds in theform of a salt with bases or acids, and the compounds in racemic form,in the form of R or S optical isomers, purified or in a mixture, offormula (I).

The present invention preferably relates to the preceding compounds offormula (I) in the D series.

There may be mentioned in particular the D-oligothionucleotide compoundproducts of general formula: ##STR19## in which J and X are defined asabove for the compounds of formula (I) and B represents a naturalnucleic base.

The oligonucleotides according to the present invention may exist in theform of homogeneous sequences, as in the compounds of formula (I) or inthe form of mixed oligomers, that is to say in the form of specificsequences included in other types of DNA or RNA type oligonucleotides,modified or otherwise, at the level of the phosphate concatenation, thelatter corresponding to compounds of formula (I), in which theintracyclic oxygen of the furanose ring is restored.

The subject of the present invention is therefore also mixed oligomericcompounds characterized in that they consist of oligothionucleotidecompounds according to the invention linked to DNA or RNA typeoligonucleotides. By "DNA or RNA type oligonucleotides" there isunderstood here a sequence of DNA or RNA type nucleic acids modified orotherwise at the level of the phosphate concatenation and in which theintracyclic heteroatom of the furanose ring of the nucleotides isoxygen.

The subject of the present invention is especially mixed oligomericcompounds consisting of an oligothionucleotide sequence according to theinvention, comprising a DNA or RNA type oligonucleotide sequence withinit or at one of its ends.

In a specific embodiment, therefore, the oligonucleotide sequence, ofthe DNA or RNA type, is flanked by two oligothioribonucleotidesequences.

The new products of general formula (I) or mixed oligomers containingthem can be prepared chemically by known processes and, in particular,those which are described in known process applications and, inparticular, those which are described in French Patent Applications FR8,310,223 (2,540,122) and FR 8,441,795 (2,568,254), WO 88/04301 and FR88 122648.

The oligothionucleotide compounds especially of formula (I) can beprepared chemically by processes in which conventional phosphodiester,phosphotriester, phosphoramidite or hydrogen phosphonate syntheses areapplied for oligonucleotides.

In these processes, the 4'-thionucleotide chain is first prepared, thevarious groups not used being protected, and then the protecting groupsare finally removed in order to obtain the desired products.

There may be mentioned in particular a process for the synthesis ofoligothionucleotide compounds according to the invention withouteffector, characterized in that a supported synthesis is carried outaccording to the phosphoroamidite method comprising

a protection in 3' and 5' of the starting 4'-thionucleotides oroligo-4'-thionucleotides with for example dimethoxytrityl in 5' andmethyl diisopropylaminophosphoramidite in 3',

the functionalization of a solid support incorporating a4'-thionucleoside derivative by for example a succinyl linkage betweenthe 3'-hydroxyl group of the 4'-thionucleoside derivative and an aminogroup of the solid support,

the elongation of the oligothionucleotide chain in a synthesizingreactor,

finally the detachment, deprotection and purification of the extendedoligothionucleotide.

There may also be mentioned a process for the synthesis ofoligothionucleotide compounds incorporating an effector according to theinvention, characterized in that there is used as starting material anoligothionucleotide without effector, or a mixed oligomer containing it,protected in 5', whose 3' OH end is reacted with an unprotectedfunctional group of a difunctional arm whose second functional group isprotected and after deprotection of the resulting product, said productis linked to the effector by the second free functional group of thedifunctional arm.

The 3' OH end of the oligomer protected in 5' can be esterified with anaminohexanoic acid, whose amine functional group is protected.

A process for the solid phase preparation of oligothionucleotidesequences according to the phoshoramidite method, which is the subjectof the present invention, comprises the following essential steps:

A 4'-thionucleoside derivative, protected by means of one of itshydroxyl functional groups in 3' or, where appropriate, 2', isimmobilized on a solid support.

The protected oligothionucleotide chain is assembled on this support, ina manual or automatic synthesizing reactor, by condensation of monomersconsisting of protected 4'-thionucleosides substituted byphosphoramidite groups in 3', where appropriate, the hydroxyl functionalgroup in 2' of the riboses being protected by Ctmp or TBDMS groups.

The oligothionucleotides are obtained after detachment of the oligomerobtained from the support and removal of the protecting groups.

Appropriately, the assembling of the oligothionucleotide is carried outby condensation, in the presence of an activating agent, of saidmonomers between their 3' functional group and the 5' functional groupof the immobilized 4'-thionucleoside compound for the first monomer orof an intermediate protected polythionucleotide compound attached tosaid immobilized thionucleoside compound for the following monomers.

In particular, the activating agent for the condensation of saidmonomers may be chosen from tetrazole and its derivatives, such aspara-nitrophenyltetrazole.

Advantageously, the phosphoramidite group in 3' of said monomers is anN,N-dialkylaminophosphoramidite group of formula ##STR20## with R₁,which represents an optionally substituted C₁ to C₇ alkyl, which may beidentical or different, such as CH₃ or (CH₃)₂ CH.

R₂ which represents an optionally substituted C₁ to C₇ alkyl, such asCH₃ or CH₂ CH₂ CN.

In particular, the phosphoramidite group in 3' of said monomers ismethyl diisopropylaminophosphoramidite (R₁ =(CH₃)₂ CH and R₂ =CH₃) or2-cyanoethyl diisopropylaminophosphite (R₁ =(CH₃)₂ CH and R₂ =CH₂ CH₂CN).

Appropriately, the hydroxyl groups of said monomers and of saidimmobilized 4'-thionucleoside compound may be protected in 5' by theDmTr group.

The immobilized 4'-thionucleoside compound may be attached to the solidsupport via a divalent succinyl group between one of its OH groups in 2'or 3' which thus becomes esterified, and an amino group of the support.

The solid support may consist of a long alkyl-amine chain attached to apolymer, a silica gel or porous glass beads.

There may be mentioned as functional group permitting the attachment orbeing linked to a solid support provided with an amino group, the groupof formula ##STR21##

with p=1 to 5, in which R₄ represents H, an activating group such as C₆Cl₅ or a radical NH linked to a solid support such as the NH radical ofan alkylamine chain attached to a polymer, silica gel or porous glassbeads.

One virtue of the homogeneous or mixed compounds according to thepresent invention, comprising an oligothionucleotide chain, is that itis possible to envisage their use independently of effectors, especiallyintercalating agents, since these new oligonucleotide substances ensurethe recognition of the complementary nucleic acid sequence, especiallyRNA sequence, with a highly increased stability; the function of theintercalating agent being to increase the stability of the hybridizationcomplex, its presence is no longer obligatory.

The compounds according to the invention, by virtue of their higholigothionucleotide sequence-specific affinity for complementary nucleicsequences, are indeed superior to the current oligonucleotides whoseaffinity for the complementary sequences is lower.

The compounds according to the present invention can therefore be usedmore efficiently, as hybridization probes, but also as purificationcomponents for given DNA or RNA sequences.

These compounds can also be used to detect mutations at the level of aDNA or an RNA more efficiently.

The compounds of the present invention, by virtue of the property of theoligothionucleotide sequences to become strongly attached to thecomplementary nucleic sequence, and then to induce the cutting of thenucleic acid chain at this site, especially when they are coupled to acutting agent, can be used as sequence-specific artificial nucleases.They can be used as reagents in molecular biology and in geneticengineering.

The presence of the hydroxyl functional group in 2', where appropriate,makes it possible, in addition, to envisage their use as artificialribozymes, whether in the form of oligothioribonucleotide sequenceswhich are homogeneous or mixed, that is to say associated with DNA orRNA type oligonucleotides.

The subject of the present invention is also the application of thecompounds according to the invention to carry out the specific blockingof cellular genes or of pathogenic agents chosen beforehand (viruses,bacteria, parasites).

The compounds according to the invention can therefore also be used asmedicinal products.

The new products of general formula (I) according to the invention andthe products of general formula (I) in which L represents an oxygenatom, R and R' each represent a hydrogen atom and B represents a naturalnucleic base, form hybridization complexes with the complementary RNAsequences which are much more stable than those which are formed withDNA.

In particular, the products of formula (I) in which J=OH(oligothioribonucleotides) pair more stably with complementary RNAsequences than the products of formula (I) in which J=H(oligothiodeoxyribonucleotides).

Given the fact that the targets for the antisense molecules in therapyare the messenger RNA genes, this property of oligothioribonucleotidesis quite useful.

This difference in the stability of the hybridization complexes permitsa preferential inhibition of the messenger RNAs and viral RNAs with nogreat risk of causing undesirable effects at the level of the DNA of thegenome.

Furthermore, with respect to the RNAs, the products according to theinvention generally form complexes which are more stable than thosewhich are obtained from natural oligonucleotides.

The oligothioribonucleotide compounds according to the invention,especially of formula (I) (J=OH), exhibit, in addition, a number ofuseful characteristics, especially for an application as antisenseagents:

the natural beta configuration is preserved,

they are isoelectric with the natural RNAs,

they have no chirality at the level of the phospho-diester bond,

they are more lipophilic than the natural analogs because of thepresence of the sulfur atom.

The sequences of the oligothionucleotides, coupled or not to anintercalating agent or to a chemically or photochemically activablegroup, comprising a concatenation of pyrimidine nucleosides are capableof attaching to a DNA or RNA double helix comprising a sequence ofadjacent purine bases associated by hydrogen bond with the complementarysequence of the pyrimidine bases. This attachment involves the localformation of a triple helix in which the pyrimidine bases of theoligothionucleotide form hydrogen bonds (Hoogsteen or reversed Hoogsteentype) with the purine bases of the double helix. In this context, theoligothionucleotides form complexes which are more stable than thecorresponding oligonucleotides and they make it possible to induceirreversible reactions (bridging, modification or cutting) on an RNA orDNA double helix.

The new products of general formula (I) according to the invention andthe products of general formula (I) in which L represents an oxygenatom, R and R' each represent a hydrogen atom and B represents a naturalnucleic base, which possess the property of attaching strongly to thecomplementary nucelic sequence, or mixed oligomers containing it, can beused as probes to detect the presence of a complementary nucleotidechain. This detection is facilitated by the presence of a fluorescentgroup or a biotin group in these molecules.

The non-natural structure of the oligothionucleotides confers propertiesof antigenicity thus rendering possible the preparation of specificantibodies directed against oligothionucleotides optionally coupled toan intercalating agent or against their complexes with a natural DNA orRNA.

For diagnostic or prognostic purposes, the oligothionucleotides,optionally coupled to an intercalating agent, or mixed oligomerscontaining them, can be covalently attached to a solid support. Thecoupling to a luminescent marker or a marker generating a colored,luminescent or fluorescent reaction makes it possible to use them asprobes for DNA or RNA sequences for diagnostic or prognostic purposes.

The oligothionucleotides according to the invention are much moreresistant to nucleases than the natural nucleoside derivatives. Thisstability with respect to enzymatic hydrolysis permits the use of theproducts according to the invention, especially of general formula (I),or mixed oligomers containing them, in experimentations in vivo or invitro in the presence of nucleases. Because of this, theoligothionucleotides according to the invention have unquestionableadvantages over the beta-nucleoside derivatives already mentioned.

The oligothioribonucleotide compounds of formula (I) for which J=OH aremore resistant to enzymatic degradation than theoligothiodeoxyribonucleotide compounds of formula I for which J=H.

Consequently, as has been seen, another subject of the present inventionrelates to the application of the new products according to theinventionk especially of general formula (I), and of the products ofgeneral formula (I) in which L represents an oxygen atom, R and R' eachrepresent a hydrogen atom and B represents a natural nucleic base, inparticular the products for which J=OH, or mixed oligomers containingthem, to the specific blocking of cellular genes or pathogenic agentschosen beforehand (viruses, bacteria, parasites), in particular mRNAs.

The new products according to the invention, especially of generalformula (I), and the products of general formula (I) in which Lrepresents an oxygen atom, R and R' each represent a hydrogen atom and Brepresents a natural nucleic base, in particular the products for whichJ=OH, or mixed oligomers containing them, are particularly useful asmedicinal products which make it possible to block the undesirableexogenous.(viruses, bacteria, parasites) or endogenous (cellular genes,oncogenes) genes, in particular the mRNAs. The expression of these genesmay be blocked by acting either directly on the DNA or RNA carrying thegenetic information, or on the messenger RNA, a copy of the gene, byblocking, in this case, any translation by hybridization or byhybridization followed by bridging or modification or cutting of themessenger or viral RNA chosen as target.

This blocking can be carried out using a product according to theinvention, especially of general formula (I), or a product of generalformula (I) in which L represents an oxygen atom, R and R' eachrepresent a hydrogen atom and B represents a natural nucleic base, inparticular the products for which J=OH, or mixed oligomers containingthem, whose sequence is complementary to that of a non-complexed regionof an RNA or a DNA and, in particular, of a messenger RNA. Thehybridization, followed or not by bridging, modification or cutting ofthe messenger or vital RNA, prevents the synthesis of RNA or of thecorresponding protein or the expression of the viral or parasiticfunctions.

If this RNA or this protein is vital for the virus, the bacterium or theparasite, the products according to the invention, especially of generalformula (I), and the products of general formula (I) in which Lrepresents an oxygen atom, R and R' each represent a hydrogen atom and Brepresents a natural nucleic base, in particular the products for whichJ=OH, or mixed oligomers containing them, will constitute medicinalproducts with antiviral, antibacterial or antiparasitic activity.

If this RNA or this protein is not vital to the organism, it is possibleto selectively suppress the effects therefrom. In this case, theproducts according to the invention, especially of general formula (I),and the products of general formula (I) in which L represents an oxygenatom, R and R' each represent a hydrogen atom and B represents a naturalnucleic base, in particular the products for which J=OH, or mixedoligomers containing them, will constitute either medicinal productswith antitumor activity when the desired gene or its messenger RNAencodes a protein involved in cell transformation, or medicinal productscapable of suppressing the character of resistance to antiviral agents,to antibiotics or to antiparasitic agents when the protein encoded isresponsible for the inactivation of the antibiotics, the antiviralagents or the anti-parasitic agents.

Specific cytotoxic effects can be obtained by action of a productaccording to the invention, especially of formula (I), or of a productof general formula (I) in which L represents an oxygen atom, R and R'each represent a hydrogen atom and B represents a natural base, inparticular the products for which J=OH, or mixed oligomers containingthem, on a cellular function essential to the target cell.

The mixed oligomers of oligothioribonucleotides according to theinvention, comprising an oligonucleotide sequence of DNA where theintracyclic oxygen is restored, are particularly valuable when used asantisense agents. These mixed oligomers are capable of being highlyselective insofar as the "window" of DNA structure is the only substrateof RNAse H after pairing of the antisense with its target complementarymRNA sequence, the said RNAse H inducing, in this case, the cutting ofthe DNA/RNA complex.

Other characteristics and advantages of the present invention willappear in the light of the following examples.

In the following description, the abbreviation rS or S_(r) precedes athioribonucleotide.

I-SYNTHESIS OF THIONUCLEOTIDES I.1- The First Syntheses of ThiosugarsWere Carried Out Between 1960 and 1970

Two examples are given:

The synthesis of derivatives of 4-thio-D-ribo-furanose is carried outaccording to scheme 1: ##STR22## This compound is obtained in 8 stepsfrom L-Lyxose with an overall yield of 6%.

R. L. WHISTLER, W. E. DICK, T. R. INGLE, R. M. ROWELL and B. URBAS. J.Org. Chem., 1964, 29, 3723-3725.

B. URBAS and R. L. WHISTLER, J. Org Chem., 1966, 31, 813-816.

E. J. REIST, D. E. GUEFFROY and L. GOODMAN, J. Am. Chem. Soc., 1964, 86,5658-5663.

R. L. WHISTLER and J. N. BeMILLER in "Methods in CarbohydrateChemistry", R. L. WHISTLER and M. L. WOLFROM, Eds., Academic Press,Inc., New York, 1962. Vol. I, p. 79.

The derivatives of 4-thio-2-deoxy-D-ribofuranose were obtained accordingto Scheme 2: ##STR23## This synthesis in 14 steps leads to methyl2-deoxy-4-thio-D-erythro-pentofuranoside with an overall yield of 8%.

Y. L. FU and M. BOBEK, J. Org. Chem. 1976, 41, 3831-3834

Y. L. FU and M. BOBEK, in "Nucleic Acid Chemistry", L. TOWSEN and R. S.TIPSON, Eds. 1978, Part 1 pp. 183-194

U. G. NAYAK and R. L. WHISTLER Liebigs Ann. Chem., 1970, 741, 131-138

Similarly, the derivatives of 4-thio-D-arabinofuranose were obtained

R. L. WHISTLER, U. G. NAYAK and W. PERKIN Jr., J. Org., Chem., 1970, 35,519-521

I.2. The corresponding nucleosides were then synthesized:

a) In 4'-thio-2'-deoxy-D-ribofuranose series

Y. L. FU and M. BOBEK in "Nucleic Acid Chemistry", L. TOWSEND, R. L.TIPSON, Eds., John Wiley & Sons, New York, 1978, p. 317.

b) In 4'-thio-D-ribofuranose series

E. J. REIST, D. E. GUEFFROY and L. GOODMAN, J. Am. Chem. Soc., 1964, 86,5658-5663.

E. J. REIST, D. E. GUEFFROY and L. GOODMAN, Chem. Ind., 1964, 1364-1365

B. URBAS and R. L. WHISTLER, J. Org. Chem., 1966, 31, 814-816

M. BOBEK, R. L. WHISTLER and A. BLOCH. J. Med. Chem., 1970, 13, 411-413

M. BOBEK, A. BLOCH, R. PARTHASARATHY and R. L. WHISTLER, J. Med. Chem.,1975, 18, 784-787.

M. BOBEK, R. L. WHISTLER and A. BLOCH, J. Med. Chem., 1972, 15, 168-171

N. OTOTANI and R. L. WHISTLER, J. Med. Chem., 1974, 17, 535-537

M. W. PICKERING, J. T. WITKOWSKI and R. K. ROBBINS, J. Med. Chem., 1976,19, 841-842.

A. K. M. ANISUZZAMAN and M. D. AMIN, J. Bangladesh Acad. Sci., 1978, 2,59-64

D. J. HOFFMAN and R. L. WHISTLER, Biochemistry, 1970, 9, 2367-2370

c) In other series:

R. G. S. RITCHIE and W. A. SZAREK, J. Chem. Soc. Chem. Commun., 1973,686

E. J. REIST, L. V. FISCHER and L. GOODMAN, J. Org. Chem., 1968, 33, 189.

R. L. WHISTLER, L .W. DONER and U. G. NAYAK, J. Org. Chem., 1971, 36,108.

These nucleosides are obtained by the traditional routes for thesynthesis of nucleosides in oxygen-containing series.

Either by the method using heavy metal salts:

Treatment of chloromercuric heterocycle with the correspondingchlorosugar

M. BOBEK, R. L. WHISTLER and A. BLOCH, J. Med. Chem., 1972, 15, 168-171

D. E. GUEFFROY and L. GOODMAN, Chem. & Ind., 1964, 1364-1365

E. J. REIST, M. BOBEK, R. L. WHISTLER and A. BLOCH, J. Med. Chem., 1970,13, 411-413

E. J. REIST, D. E. GUEFFROY and L. GOODMAN, J. Am. Chem. Soc., 1964, 86,5658-5663.

E. J. REIST, L. V. FISCHER and L. GOODMAN, J. Org. Chem., 1968, 33,189-192

Or by the HILBERT and JOHNSON method,

by condensation of the pyrimidine base with the correspondingthiochlorosugar

B. URBAS and R. L. WHISTLER, J. Org. Chem., 1966, 31, 813-816.

by condensation of the silylated derivatives of the bases and thecorresponding thiochlorosugars

M. BOBEK, A. BLOCH, R. PARTHASARATHY and R. L. WHISTLER, J. Med. Chem.,1975, 18, 784-787

R. L. WHISTLER, L. W. DONER and U. G. NAYAK, J. Org. Chem., 36, 108-110

N. OTOTANI and R. L. WHISTLER, J. Med. Chem., 1974, 17, 535-537.

By the reaction of fusion of 3-cyano-1,2,4-triazole and1,2,3,5-tetra-O-acetyl-4-thio-D-ribo-furanose.

M. V. PICKERING, J. T. WITKOWSKI and R. K. ROBINS, J. Med. Chem., 1976,19, 841-842.

By the VORBRUGGEN and NIEDBALLA method by condensation of adenine and1,2,3,5-tetra-O-acetyl thioribofuranose in the presence ofFRIEDEL-CRAFTS catalyst.

A. K. M. ANISUZZAMAN and M. D. AMIN, J. Bangladesh Acad. Sci., 1978. 2,59-64

Recently, the synthesis of thionucleosides has again attracted interest

Thus, 2',3',5'-tri-O-benzyl-4'-thio-β-D-xylofuranosyl uracil wasobtained by a novel route according to Scheme 3:

M. W. BREDENKAMP, C. W. HOLZAPFEL and A. D. SWANEPOEL, Tetrahedron Lett.1990, 31, 2759-2762.

M. W. BREDENKAMP, C. W. HOLZAPFEL and A. D. SWANEPOEL, S. Atr. J. Chem.1991, 44. 31-33. ##STR24## The synthesis of pyrimidine analogs of4'-thio-2'-deoxy-nucleosides has been published

M. R. DYSON, P. L. COE and R. T. WALKER, J. Med. Chem., 1991, 34,2782-2786. by the HORTON and MARKOVS method.

D. HORTON and R. A. MARKOVS, Carbohydrate Res., 1980, 80, 356.

It is illustrated by Scheme 4: ##STR25## Similarly,2'-deoxy-4'-thiocytidine, 2'-deoxy-4'-thiouridine and 4'-thiothymidinewere obtained by J. A. SECRIST III et al., (J. A. SECRIST, K. N. TIWARI,J. M. RIORDAN and J. A. MONTGOMERY, J. Med. Chem., 1991, 34, 2361-2366);(J. A. MONTGOMERY and J. A. SECRIST III. PCT Int. Appl. WO 9104,033)from 2-deoxy-4-thio-β-D-erythropentofuranose synthesized according toBOBEK et al., (Y. L. FU, M. BOBEK, J. Org. Chem., 1976, 41, 3831-3834)using TMSTf as coupling catalyst.

The synthesis of 4'-thio-2'-deoxy-D-ribofuranose has been the subject ofa patent and publications

European Patent Application, 0421 777 Al, R. T. WALKER.

M. R. DYSON, P. O. COE and R. T. WALKER, J. Chem. Soc. Chem. Comm.,1991, 741-742.

M. R. DYSON, P. L. COE and R. T. WALKER, Carbohydrate Res., 1991, 216,237-248

It is described in Scheme 5: ##STR26## Ribothionucleosides derived frompyrimidines were the subject of the same patent. But the synthesis ofthe starting thioribofuranose was carried out according to the method ofE. J. REIST.

E. J. REIST, D. E. GUEFFROY and L. GOODMAN, J. Am. Chem. Soc., 1964, 84,5658

I.3. SYNTHESIS OF SYNTHONS OF 4'-THIO-BETA-D-RIBOFURANOSE NECESSARY FORTHE PRODUCTION OF THIOOLIGOMERS

The synthesis of L-thioLyxofuranose and D-thioRibofuranose was carriedout in 7 steps from D-ribose and L-lyxose respectively with 29 and 21%yield respectively. This synthesis is illustrated in Scheme 6. ##STR27##The synthesis in order to obtain the thio-D-ribofuranose uses L-lyxoseas starting product as in the Whistler synthesis (page 19). Thisstrategy is called strategy C1-C4. Indeed, the sulfur atom is firstintroduced in the anomeric position and an NS2 nucleophilic substitutionbetween the sulfur atom carried by the anomeric carbon (C1) and the C4carbon carrying an OH activated by a Mesyl (Ms) group is then carriedout. This strategy applied to the synthesis of 4-thio-D-ribofuranose and4-thio-L-lyxofuranose resembles that which Walker used to obtain the2-deoxy-D-ribofuranose series (Patent Eur. Pat. 0421 777 A 1 page 6).For example, WALKER's product 5 (Scheme 5) is similar to our product 10(Scheme 6) in L-lyxofuranose series. ##STR28## The synthesis of thethionucleosides of uracil, thymine and cytosine was carried out usingbis-trimethylsilyl acetamide (BSA) as silylating agent andtrimethylsilyl trifluoromethanesulfonate (TMSTf) as coupling agent. TheO and β nucleosides were obtained with 74, 77 and 70% yieldsrespectively. The synthesis of the thionucleosides of adenine wascarried out with SnCl4 in acetonitrile. The O and β nucleosides areobtained with 58% yield (Scheme 7).

II. SYNTHESIS OF OLIGOTHIONUCLEOTIDES ON A SOLID SUPPORT

The supported synthesis of β oligothioribonucleotides was carried outwith the aid of an APPLIED BIOSYSTEM 318A model GENE synthesizeraccording to the phosphoramidite method.

It is similar to that described for the natural βoligodeoxyribonucleotide series by CARUTHERS et al., (S. L. BEAUCAGE andM. H. CARUTHERS, Tetradedron Lett., 1981, 22, 1859-1862; L. J. MCBRIDEand M. H. CARUTHERS, Tetrahedron Lett., 1983, 24, 245-248.)

and to that described by USMAN et al., for the oligoribonucleotides (N.USMAN, K. K. OGILVIE, M. Y. JIANG and R. J. CEDERGREN, J. Am. Chem.Soc., 1987, 109, 7845-7854.).

This synthesis required:

I) The preparation of synthons β thioribonucleoside phosphoramidites 5corresponding to the bases T, U, C, A and G.

II) The functionalization of the solid support incorporating a βthioribonucleoside derivative.

III) The elongation of the oligothioribonucleotide chain by means of anautomatic synthesizer.

IV) Finally, at the end of the required number of synthesis cycles, theoligothionucleotide was detached from its support, deprotected andpurified.

II.1--Synthesis of the Phosphoramidite of the Beta-thioribonucleoside 26a-d. II.1.1 Synthesis of 5'-DmTr-2'-O-TBDMS Derivatives (Scheme 8)

The major problem in the chemical synthesis of the oligoribonucleosidesis the presence of hydroxyl in 2' in the ribose or thioribose ring whichrequires a selective protection.

The selection of a protecting group for the hydroxyl in 2' shouldcorrespond to several criteria; it should be stable:

under the coupling conditions during the deprotection of the protectinggroups in 5' to allow the extension of the chain,

during the oxidation in the case of the phosphite triester method,

during the masking which follows the coupling,

during the deprotection of the exocyclic amine-protecting groups

during the deprotection of the phosphates,

and in the case of the syntheses on a solid support during the cleavageof the oligomer from the support.

Finally, this protecting group should be removed under very mildconditions to avoid an attack of the liberated hydroxyl functional groupin 2' on the adjacent phosphodiester bond.

A large number of strategies for protecting the hydroxyls in 2' havebeen developed and illustrate the difficulties in the choice of thescheme for protection of the hydroxylated functional groups (C. B.REESE, Nucleosides Nucleotides, 1985, 4, 117-127; R. KIERZECK, M. H.CARUTHERS, C. E. LONGFELLOW, D. SWINTON, D. H. TURNIER and S. M. FREIER,Biochemistry, 1986, 25, 7840-7846; S. IWAI and E. OHTSUKA, Nucleic AcidsRes., 1988, 16, 9443-9456; C. LEHMANN, Y. Z. XU, C. CHRISTODOULOU, Z. K.TAN and M. J. GAIT, Nucleic Acids Res. 1989, 17, 2379-2390; T. TANAKA,S. TAMATSUKURI and M. IKEHARA, Nucleic Acids Res.. 1986, 14,6265-6279.).

The use of alkylsilyl protecting groups and especiallytert-butyldimethylsilyl (TBDMS) for the hydroxyl in 2' has facilitatedthe synthesis of oligoribonucleotides. The TBDMS group is sufficientlystable under acidic or basic conditions and is easily removed bytetra-butyl ammonium fluoride in THF for use in the solid phase strategy(N. USMAN, K. K. OGILVIE, M. Y. JIANG and R. L. LEDERGEN, J. Am. Chem.Soc., 1987, 109, 7845-7854; N. USMAN, K. NICOGHOSIAN, R. L. CEDERGRENand K. K. OGILVIE, Proc. Natl. Sci. USA, 1988, 85, 5764-5768; S. A.SCARINGE, C. F. FRANCKLYN and N. USMAN, Nucleic Acids Res., 1990, 18,5433-5441; K. K. OGILIVIE and M. J. NEMER, Tetrahedron Lett., 1980, 21,4159; R. T. PON and K. K. OGILVIE, Nucleosides Nucleotides, 1984, 3,485; R. T. PON and K. K. OGILVIE, Tetrahedron Lett., 1984, 25, 713).

The protection scheme used for the oxygen-containing nucleosides (Scheme8) was therefore applied in β oligothioribonucleotide series

Namely the following protections:

benzoyl for adenine and cytosine, isoButyl for guanine as protection forthe exocyclic amino groups,

dimethoxytrityl (DmTr) to protect the primary hydroxyls in 5',

tert-butyldimethylsilyl (TBDMS) to protect the secondary hydroxyl in 2',

methoxy for the protection of the phosphates.

The first step makes it possible to protect the free hydroxyl in 5' by alabils acidic group DmTr (Scheme 8). Thus, the nucleoside derivative 22is treated with dimethoxytrityl chloride (1.27 molar equivalents) inanhydrous pyridine under an inert atmosphere. After the usual treatmentof the reaction mixture, the dimethoxytritylated derivative 23 ispurified by silica gel chromatography and isolated with yields rangingfrom 70 to 80%.

The physico-chemical characteristics of these derivatives 23 (NMR, mass)are in agreement with the proposed structures. ##STR29##

The second step (Scheme 8) consists in protecting the secondary hydroxylin 2'. However, during the silylation, a mixture of 2'-O-silyl 24 andits regioisomer 3'-O-silyl 25 is obtained (K. K. OGILVIE and D. W.ENTWISTLE, Carbohyd. Res., 1981, 89, 203-210, K. K. OGILVIE, A. L.SCHIFMAN and C. L. PENNEY, Can. J. Chem., 1979, 57, 2230, G. H.HAKIMELAHI, Z. A. PROBA and K. K. OGILVIE, Can. J. Chem., 1982, 60,1106). These isomers should be separated with great care on a silica gelcolumn in order to obtain the 2'-O-TBDMS 24 derivative with a puritygreater than 99.95% measured by HPLC. Indeed, if this is not the case,the subsequent steps will lead us to the expected mixtures of 5'-3'oligothionucleotide with the undesirable 5'-2' isomer. Consequently, thepurity of the 5'-O-DmTr-2'-O-silyl 24 is of prime importance for a goodsynthesis.

Thus, the production of the compounds 5'-DmTr-2'-O-TBDMS 24 was firstcarried out according to the method of OGILVIE et al., (K. K. OGILVIE,A. L. SCHIFMAN and C. L. PENNEY, Can. J. Chem., 1979, 57, 2230) whichconsists in condensing 23 with tert-butyldimethylsilyl chloride(TBDMSCl) (1.2 molar equivalent) in pyridine in the presence ofimidazole (2.6 molar equivalents). At the end of the reaction, a mixtureof 2'-O-TBDMS derivative 24 and its 3'-O-TBDMS isomer 25 is obtained.The expected compound 24 is separated from the mixture by chromatographyon a silica gel column.

It is then possible, by an equilibration reaction, to obtain 24 from its3'-O-TBDMS isomer 25 (S. S. JONES and C. B. REESE, J. Chem. Soc. PerkinTrans I, 1979, 2762).

This migration of the TBDMS group is intramolecular and occurs via anintermediate possessing a pentavalent silicon atom. The isomerization ofthe 3'-O-TBDMS derivative previously obtained after a firstchromatographic separation is carried out in ethanol at room temperatureovernight. The two position isomers thus obtained are again separated bychromatography on a silica gel column. This isomerization operationfollowed by a purification is repeated three times.

However, the 5'-DmTr-2'-O-TBDMS 24 yields obtained by this method (40 to50% according to the bases), did not appear to us to be satisfactory. Weused another more recent method developed by OGILVIE (G. H. HAKIMELAHI,Z. A. PROBA and K. K. OGILVIE, Can. J. Chem., 1982, 60, 1106) whichpermits the preferential introduction of TBDMS in postion 2' of the βribonucleosides. This technique recommends the use of silver salts.

Thus, 5'-dimethoxytrityl 23 is condensed with TBDMS chloride (1.3 molarequivalents) in the presence of silver nitrate (1.2 molar equivalents)and 3.7 equivalents of pyridine in anhydrous THF. After purification bychromatography on a silica gel column, 56% yield of 24 and 27% of5'-DmTr-3'-O-TBDMS 25 are obtained. Consequently, 5'-DmTr-2'-O-TBDMS 24is obtained under these new conditions in a single step with a betteryield but the selectivity of introduction of the TBDMS group in 2' isnot improved.

II.1.2. Synthesis of the Phosphoramidite Derivatives 26 a-d (Scheme 9)

The four N-protected β thioribonucleoside phosphoramidites 26 a-d arethen obtained from the corresponding N-protected 5'-DmTr-2'-O-TBDMS βthioribonucleosides 24 (Scheme 9).

The nucleoside derivative 24 was treated withchloro-N,N-diisopropylaminomethoxyphosphine (2.5 molar equivalents) inthe presence of N,N,N-diisopropylethylamine (DIEA) in dichloromethaneunder an inert atmosphere. The phosphoramidite derivative 26 waspurified by silica gel chromatography and freeze-dried in benzene. Thephosphoramidites 26 a-d are obtained with yields ranging from 78 to 80%depending on the nature of the base. ##STR30## Analyses of the ³¹ P and¹ H NMR spectra of 26 show clearly that a single pair ofdiastereoisomers is obtained, which establishes the regioisomeric purityof the compounds obtained.

Indeed, some authors (R. KIERZEK, M. H. CARUTHERS, C. E. LONGFELLOW, D.SWINION, D. H. TURNER and S. M. FREIER, Biochemistry, 1986, 25,7840-7846) had emitted the hypothesis that the silylated groups inposition 2' were not stable under the phosphorylation conditions.However, it was shown (W. K. KOHLER, W. SCHLOSSERM, G. CHARUBALA and W.PFLEIDLERER, Chemistry and Biology of Nucleosides and Nucleotides,Academic Press, New York, 1978, pp. 347-358; K. K. OGILVIE and D. W.ENTWISTLE, Carbohydrate Res., 1981, 89, 203-210; N. USMAN, K. K.OGILVIE, M. Y. JIANG and R. J. CEDERGREN, J. Am. Chem. Soc., 1987, 109,7845-7854) that the alkylsilyl groups migrate in protic solvents such asmethanol or in aqueous solutions such as a pyridine/water mixture butthese silylated groups are stable in anhydrous solvents and especiallyin non-protic bases such as anhydrous pyridine (K. K. OGILVIE, D. W.ENTWISTLE, Carbohydrate Res., 1981, 89, 203-210; W. KOHLER, W.SCHLOSSER, G. CHARUBALA and W. PFLEIDERER, Chemistry and Biology ofNucleosides and Nucleotides, Academic Press, New York, 1978, pp.347-358). Moreover, it has been clearly demonstrated in ribonucleotideseries that the use of the 2'-O-silyl ribonucleosides in the synthesisof oligoribonucleotides lead exclusively to 5'-3' bonds (K. K. OGILVIE,M. J. NEMER, Tetrahedron Lett., 1980, 21, 4159; R. T. PON and K. K.OGILVIE, Tetrahedron Lett., 1984, 25, 713; R. T. PON and K. K. OGILVIE,Nucleosides Nucleotides, 1984, 3, 485; P. J. GAREGG, I. LINDH, J.STAWINSKI and R. STOMBERG, Tetrahedron Lett., 1986, 27, 4055-4058). Thesynthesis of phosphoramidites is not stereospecific and the formation inequal quantities of these two disasteroisomers is due to the chiralityof the phosphorus atom of R or S configuration. The formation of thesetwo products is not disruptive insofar as the subsequent oxidationreactions suppress this chirality.

II.2. EXPERIMENTAL PART General Method for the Preparation of the5'-O-dimethoxy-trityl-N-acyl-4'-thio-β-D-ribonucleosides 23 a-d.

Dimethoxytrityl chloride (1.27 mmol, 430 mg) is added to a solution ofN-acyl-β-D-thioribonucleoside 23 a-d (1 mmol) in anhydrous pyridine (5.6ml). The reaction mixture is stirred at room temperature under an inertatmosphere for 90 to 120 min and then methanol (1 ml) is added. Afterstirring for an additional 10 minutes, the reaction mixture is pouredover a saturated aqueous sodium bicarbonate solution (25 ml) and theproducts are extracted with dichloromethane (2×20 ml). The organicphases are washed with water (2×100 ml) and then dried over sodiumsulfate and filtered. The filtrate is evaporated under reduced pressureand the residue obtained is chromatographed on a silica gel column. Theelution is carried out with a CH₂ Cl₂ /MeOH mixture: 98/2 in thepresence of 1% triethylamine; the fractions containing the product areevaporated to dryness and the nucleosides 23 a-d are obtained in theform of an oil.

1-[4'-thio-5'-O-dimethoxytrityl-β-D-ribofuranosyl]Thymine 23 a.

Yield: 68%

Mass spectrometry FAR>0 NOBA m/z=577 [M+H]+, 303 [DmTr]+

¹ H NMR (DMSOd₆, 360 MHz) δ11.24, (s, 1 H, NH); 7.44, (s, 1 H, H₆);6.87-7.30, (m, 13 H, H Aromatic); 5.83, (d, 1 H, H_(1'), J_(1'),2'=7.0); 5.53, (d, 1 H, OH_(2'), J_(OH),2' =5.8); 5.30 (d, 1 H, OH_(3'),J_(OH),3' =4.7); 4.14 (dd, 1 H H_(2'), J_(2'),1' =6.9, J_(2'),3' =3.0);4.00, (t, 1 H, H_(3'), J_(3'),2' =3.0, J_(3'),4' =3.0); 3.69, (s, 6 H,OMe); 3.30, (dd, 1 H, H_(4')); 3.13, (m, 2 H, H_(5'), H_(5")); 1.60, (s,3 H, CH₃).

1-[4'-thio-5'-O-dimethoxytrityl-β-D-ribofuranosyl]Uracil 23 b.

Yield: 70%

¹ H NMR (DMSOd₆, 360 MHz) δ11.31, (s, 1 H, NH); 7.70, (d, 1 H, H₆, J₆.5=8.0), 7.40-6.89, (m, 13 H, aromatic H of the dmTr group ); 5.84, (d, 1H, H_(1'), J_(1'),2' =5.7), 5.53, (d, 1 H, OH_(2'), J_(OH2'),2' =5.6),5.48, (d, 1 H, H₅, J₅.6 =8.0); 5.26, (d, 1 H, OH_(3'), J_(OH3'),3'=4.45); 4.02, (m, 2 H, H_(2'), H_(3')); 3.74, (s, 6 H, OMe); 3.31, (m, 3H, H_(4'), H_(5'), H_(5"))

Mass spectrometry FAB>0 NOBA m/z=563 [M+H]+, 303[DmTr]+1-[4'-thio-5'-O-dimethoxytrityl-β-D-ribofuranosyl]N-4-benzoylCytosine23 c.

Yield 69%

¹ H NMR (DMSOd₆, 250 MHz) δ11.32, (s, 1 H, NH); 8.40, (d, 1 H, H₆, J₆.5=7.5); 8.01, (d, 2H, Ho of the benzoyl group); 7.63, (d, 1H, H₅, J₅.6=7.3); 7.44, (m, 17H, aromatic H of the DmTr group and Hm,p of thebenzoyl group); 5.88, (d, 1H, H_(1'), J_(1'),2' =4.1); 5.73 (d, 1H,OH_(2'), J_(OH),2' =5.2); 5.27 (d, 1H, OH_(3'), J_(OH),H3' =5.7); 4.06(m, 1H, H_(2'), J_(2'),1' =4.0, J_(2'),3' 32 3.7, J_(2'),OH =5.2); 4.03,(m, 1H, H_(3'), J_(3'),2' =3.7, J_(3'),4' =5.3, J_(3'),OH =5.7); 3.76,(s, 6H, OCH₃ of the DmTr group); 3.43, (m, 3H, H_(4'), H_(5') andH_(5")).

Mass spectrometry FAB>0 NOBA m/z=666 [M+H]+, 517 [M+H.o slashed.CONH₂]+, 303 [DmTr]+

1-[4'-thio-5'-O-dimethoxytrityl-β-D-ribofuranosyl]N6-benzoylAdenine 23d.

Yield 70%

¹ H NMR (DMSOd₆, 250 MHz) δ11.25, (s, 1 H, NH); 8.63, (s, 1H, H₈); 8.57,(s, 1H, H₂); 8.04, (d, 2H, Ho of the benzoyl group); 7.51, (m, 13H,aromatic H of the DmTr group); 6.92, (d, 3H, Hm,p of the benzoyl group);5.98, (d, 1H, H_(1'), J_(1'),2' =5.8); 5.75, (d, 1H, OH_(2'), J_(OH),2'=3.5); 5.45, (d, 1H, OH_(3'), J_(OH),3' =2.5); 4.70, (m, 1H, H_(2'));4.26, (m, 1H, H_(3')); 3.74, (s, 6H, OCH₃ of the DmTr group); 3.53, (m,3H, H_(4'), H_(5') and H_(5"))

Mass spectrometry FAB>0 NOBA m/z=690 [M+H]+, 303 [DmTr]+

General method for the preparation of the2'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-N-acyl-β-D-ribonucleosides24 a-e and their 3'-TBDMS isomer 25 a-e.

Silver nitrate (1.2 mmol, 203 mg) is added to a solution of5'-O-dimethoxytrityl-4'- thio-N-acyl-β-D-ribonucleoside 23 a-d (1 mmol)in anhydrous THF (10 ml). The reaction mixture is then stirred for 5minutes at room temperature before adding tert-butyldimethylsilylchloride (TBDMSCl, 1.3 mmol, 195 mg) and anhydrous pyridine (3.7 mmol,0.193 ml). The stirring at room temperature is then continued for 24 hand then the beterogenous solution is filtered before being poured intoa 5% aqueous sodium bicarbonate solution (30 ml). The products areextracted with dichloromethane (3×20 ml) and the organic phases arewashed with water and then dried over sodium sulfate and filtered. Theliltrate is evaporated to dryness and the residue is chromatographed ona silica gel column using as eluent a CH₂ Cl₂ /AcOEt mixture: 95/5 andthen with a CH₂ Cl₂ /AcOEt mixture: 80/20. The fractions containing thedesired product (2'-O-TBDMS, 24 a-d) are pooled and evaporated todryness. The 3'-O-TBDMS isomer 25 a-d is then obtained.

1 -[2'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-β-D-ribofuranosyl]Thymine 24 a.

yield: 33%

Mass spectrometry FAB>O NOBA m/z=691 [M+H]+, 713 [M+Na]+

¹ HNMR (DMSOd₆, 300 MHz) δ11.36, (s, 1 H, NH); 7.58, (s, 1H, H₆);7.33-6.90, (m, 13H, aromatic H); 5.89, (s, 1H, H_(1'), J_(1'),2' =6.1);5.27, (m, 1H, OH_(3')); 4.20, (dd, 1H, H, H_(2'), J_(2'),1' =6.10,J_(2'),3' =3.5); 3.97, (m, 1H, H_(3')); 3.73, (s, 6H, OMe); 3.36, (m,1H, H_(4')), 3.25, (m, 2H, H_(5'), H_(5")); 1.58, (s, 3H, CH₃); 0.81,(s, 9H, tert-butyl); 0.0 (d, 6H, Me₂ Si).

1-[2'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-β-D-ribofuranosyl]Uracil24 b.

Yield: 56%

m.p.=114° C.

¹ H NMR (DMSOd₆, 360 MHz) δ11.20, (s, 1 H, NH); 7.81, (d, 1H, H₆, J₆,5=8.1); 7.30-6.89, (m, 13H, aromatic H of the dmTr group) 5.84, (d, 1H,H_(1'), J_(1'),2' =5.5); 5.50, (d, 1H, H₅, J₅,6 =8.1); 5.23, (d, 1H,OH_(3'), J_(OH3'),3' =4.7); 4.11, (q, 1H, H_(2'), J_(2'),1' =5.5,J_(2'),3' =3.5); 3.95, (m, 1H, H_(3')); 3.74, (s, 6H, OMe); 3.38, (m,2H, H_(4'), H_(5'), J_(4').5' =3.28, (m, 1H, H_(5")); 0.82, (s, 9H,tert-butyl); 0.05, (m, 6H, Me₂ Si).

Mass spectometry FAB>0 NOBA m/z=677 [M+H]+, 619 [M+H-tert-butane]+, 303[dmTr]+

1-[2'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-β-D-ribofuranosyl]-N4-benzoylCytosine24 c.

Yield: 32%

¹ H NMR (DMSOd₆, 250 MHz): δ11.31, (s, 1 H, NH); 8.51, (d, 1H, H₆, J₆.5=7.3); 8.01, (d, 2H, Ho of the benzoyl group); 7.51, (m, 14H, aromatic Hof the DmTr group and H₅); 6.93, (d, 3Hm,p of the benzoyl group); 5.93,(d, 1H, H_(1'), J_(1'),2' =3.0); 5.20, (d, 1H, OH_(3'), J_(OH),H3'=5.0); 4.14, (m, 1H, H_(2')); 4.00, (m, 1H, H_(3')); 3.76, (s, 6H, OCH₃of the DmTr group); 3.38, (m, 3H, H_(4'), H_(5') and H_(5")); 0.86, (s,9H, tert-butyl); 0.60, (d, 6H, Me₂ Si).

Mass spectrometry FAB>0 NOBA m/z 780 [M+H]⁺, 303 [DmTr]⁺

1-[2'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-β-D-ribofuranosyl]-N6-benzoylAdenine24 d.

Yield: 35%

¹ H NMR (DMSOd₆, 250 MHz): δ11.25, (s, 1 H, NH); 8.64, (s, 1H, H₈);8.61, (s, 1H, H₂) , 8.04, (d, 2H, Ho of the benzoyl group); 7.51, (m,13H, aromatic H of the DmTr group); 6.95, (d, 3H, Hm,p of the benzoylgroup); 6.01, (d, 1H, H_(1'), J_(1'),2' =5.8); 5.48, (d, 1H, OH_(3'),J_(OH),3' =2.5); 4.70, (m, 1H, H_(2')); 4.26, (m, 1H, H_(3')); 3.75, (s,6H, OCH₃ of DmTr group); 3.53, (m, 3H, H_(4'), H_(5') and H_(5")); 0.82,(s, 9H, tert-butyl); 0.50, (d, 6H, Me₂ Si).

Mass spectometry FAB>0 NOBA m/z=804 [M+H]+, 303 [M+H]+

1-[3'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-β-D-ribofuranosyl]Thymine 25 a.

Yield: 13%

¹ H NMR (DMSOd₆, 360 MHz): δ11.34, (s, 1 H, NH); 7.46, (s, 1H, H₆);7.89-7.30, (m, 13H, aromatic H); 5.87, (d, 1H, H_(1'), J_(1'),2' =7.8);5.46, (d, 1H, OH_(2'), J_(OH),2' =5.1); 4.13, (m, 1H, H_(3')); 4.11, (m,1H, H_(2'), J_(2'),1' =7.8, J_(OH),2' =5.1); 3.73, (s, 6H, OMe); 1.66,(s, 3H, CH₃); 0.84, (s, 9H, tert-butyl); 0.50, (d, 6H, Me₂ Si).

1 - [3'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-β-D-ribofuranosyl]Uracil 25 b.

Yield: 27%

m.p.=109° C.

¹ H NMR (DMSO₆, 360 MHz): δ11.30, (s, 1 H, NH); 7.70, (d, 1H, H₆, J₆,5=8.0); 7.30-6.89, (13 H, aromatic H of the dmTr group); 5.84, (d, 1 H,H_(1'), J_(1'),2' =7.3); 5.57, (d, 1 H, H₅, J₅,6 =8.0); 5.46, (d, 1 H,OH_(2'), J_(OH2'),2' =5.1); 4.12, (t, 1 H, H_(3'), J_(3'),2' =3.1,J_(3'),4' =3.1); 4.03, (m, 1H, H_(2')); 3.74, (s, 6H, OMe);3.41, (m, 1H,H_(4')); 3.21, (m, 2H, H_(5'), H_(5")); 0.08, (s, 9H, tert-butyl); 0.02(m, 6H, Me₂ Si).

Mass spectrometry FAB>0 NOBA m/z=677 [M+H]+, 619 [M+H-tert-butane]+, 303[dmTr]+.

This compound was brought into contact with a 0.25% (v/v)ethanol/triethylamine solution for 12 h at room temperature in order togive, in equal proportions, a mixture of 24 b and 25 b.

1-[3'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-β-D-ribofuranosyl]-N4-benzoylCytosine25 c.

Yield: 31%

¹ H NMR (DMSOd₆, 250 MHz): δ11.33, (s, 1 H, NH); 8.40, (d, 1H, H₆, J₆,5=7.4); 8.01, (d, 2H, Ho of the benzoyl group); 7.60 (m, 14H, aromatic Hof the DmTr group and H₅); 6.92, (d, 3H, Hm,p of the benzoyl group);5.92, (d, 1H, H_(1'), J_(1'),2' =5.3); 5.60, (d, 1H, OH_(2'), J_(OH),2'=5.0); 4.15, (m, 2H, H_(2'), H_(3')); 3.75 (s, 6H, OCH₃ of the DmTrgroup); 3.38, (m, 3H, H_(4'), H_(5'), and H_(5")); 0.79, (s, 9H,tert-butyl); 0.00, (d, 6H, Me₂ Si)

Mass spectrometry FAB>0 NOBA m/z 780 [M+H]⁺, 303 [DmTr]⁺

1-[3'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-β-D-ribofuranosyl]-N6-benzoylAdenine25 d.

Yield: 29%

¹ H NMR (DMSOd₆, 250 MHz): δ11.25, (s, 1 H, NH); 8.64, (s, 1H, H₈);8.61, (s, 1H, H₂); 8.04, (d, 2H, Ho of the benzoyl group); 7.51, (m,13H, aromatic H of the DmTr group); 6.95, (d, 3H, Hm,p of the benzoylgroup); 6.01, (d, 1H, H_(1'), J_(1'),2' =5.8); 5.79, (d, 1H, OH_(2'),J_(OH),2' =3.5); 4.70, (m, 1H, H_(2')); 4.26, (m, 1H, H_(3')); 3.75, (s,6H, OCH₃ of the DmTr group); 3.53, (m, 3H, H_(4'), H_(5') and H_(5"));0.82, (s, 9H, tert-butyl); 0.01, (d, 6H, Me₂ Si).

Mass spectrometry FAB>0 NOBA m/z 804 [M+H]⁺, 303 [M+H]⁺

General method for the preparation of the β-D-ribo-nucleosidephosphoramidites 26 a-d.

N,N,N-diisopropylethylamine (4 mmol, 0.7 ml),chloro-N,N-diisopropylaminomethoxyphosphine (2.5 mmol), 0.48 ml) and4-N,N-dimethylaminopyridine (0.2 mmol, 24.4 mg) are added under an inertargon atmosphere to a solution of the 24 a-d compounds (1 mmol) inanhydrous dichloromethane (3.8 ml). The reaction mixture is stirred for40 to 90 minutes at room temperature and is then diluted with ethylacetate (35 ml). The resulting solution is washed with brine (4×50 ml)and then with water (2×50 ml). The organic phase is dried over sodiumsulfate, filtered and evaporated under reduced pressure. The residue ischromatographed on a silica gel colunm and the elution is carried out bymeans of a mixture of cyclohexane, dichloromethane and triethylamine(100/0/0.1 to 50/50/0.1). The 26 a-d products are obtained in the formof a white powder after freeze-drying in benzene.

1-[2'-O-tert-butyldimethylsilyl-3'-N,N-diisopropylmethoxyphosphoramidite-4'thio-5'-O-dimethoxytrityl-β-D-ribofuranosyl]Thymine26 a (diastereoisomeric mixture)

Yield: 89%

³¹ P NMR (CD₃ CN): δ151.32 and 150.03.

¹ H NMR: δ7.99, (s, 1 H, NH); 7.65, (d, 1 H, H₆); 7.35-6.80, (m, 13 H,aromatic H); 5.97, (m, 1 H, H_(1')); 4.25, (m, 1 H, H_(2')); 4.13, (m,1H, H_(3')); 3.78, (s, 6 H, OMe); 3.68, (m, 1 H, H_(4')); 3.54, (m, 2 H,H_(5'), H_(5")); 3.35 (m, 3 H, MeO of the phosphoramidite group); 3.25,(m, 2 H, CH of the isopropyl groups); 1.60, (m. 3 H, CH₃); 1.15, (m, 12H, CH₃ of the isopropyl groups); 0.86, (s, 9 H, tert-butyl); 0.05, (m, 6H, Me₂ Si); mass spectrometry FAB>0 NOBA m/z=852 [M+H]⁺

1-[2'-O-tert-butyldimethylsilyl-3'-N,N-diisopropylmethoxyphosphoramidite-4'-thio-5'-O-dimethoxytrityl-β-D-ribofuranosyl]Uracil26 b (diastereoisomeric mixture)

Yield: 78%

³¹ P NMR (CDCl₃): δ150.65 and 150.50.

¹ H NMR: (CD₃ CN, 250 MHz): δ7.99, (s, 1 H, NH); 7.22, (m, 1 H, H₆);7.30-6.89, (m, 13 H, aromatic H of the DmTr group); 5.90, (m, 1H,H_(1')); 5.50, (m, 1 H, H₅); 4.13, (m, 2 H, H_(2'), H_(3')); 3.80, (s, 6H, OMe of group DmTr); 3.58, (m, 3 H, H_(4'), H_(5'), H_(5")); 3.42, (m,2 H, 2 CH of isopropyl groups); 3.30, (m, 3 H, MeO of the DmTr group);1.19, (m, 12 H, CH₃ of isopropyl groups); 0.84, (s, 9 H, tert-butyl);0.05, (m, 6 H, Me₂ Si).

Mass spectrometry FAB>0 NOBA m/z=534 [M+H-DmTrH]⁺

1-[2'-O-tert-butyldimethylsilyl-3'-N,N-diisopropylmethoxyphosphoramidite-4'thio-5'-O-dimethoxytrityl-β-D-ribofuranosyl]-N4-benzoylCytosine26 c.

Yield: 75%

³¹ P NMR (CD₃ CN): δ151.30 and 150.05.

Mass spectrometry FAB>O PEG m/z 941 [M+H]⁺, 637 [M+H-DmTrH], 303 [DmTr]⁺

1-[2'-O-tert-butyldimethylsilyl-3'-N,N-diisopropyl-methoxyphosphoramidite-4'thio-5'-O-dimethoxytrityl-β-D-ribofuranosyl]-N6-benzoylAdenine26 d.

Yield: 71%

³¹ P NMR (CDCl₃): δ151.26 and 150.01

Mass spectrometry FAB>O PEG m/z 964 [M+H]⁺, 600 [M+H-DmTrH], 303 [DmTr]⁺

II.3 Functionalization of the Solid Support (Scheme 10)

The solid support or LCA-CPG (Long Chain Alkylamine Controlled PoreGlass) P-1 was first activated by a 3% trichloroacetic acid solution indichloromethane at room temperature for 2 to 3 hours in order toliberate the highest number of amino groups, which leads to the maximumnumber of reactive sites on the surface of the glass beads. The thusactivated support P-2 is then functionalized by coupling with succinicanhydride in pyridine in the presence of 4-DMAP, which leads to P-3.

In the following step, the 3'-silylated nucleoside 25 is used since itwas obtained during the reaction for silylation of thethioribonuleosides and is not necessary for the synthesis ofoligothionucleotides containing the 3'-5' phosphodiester bond. Thus, P-3activated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (DEC) in thepresence of 4-DMAP is reacted in anhydrous pyridine with5'-O-DmTr-3'-OTBDMS-β-D-thioribonucleoside 25. The unused sites of thesupport are masked by means of piperidine. A final step of masking withacetic anhydride in the presence of collidine in THF leads to P-6. Thequantity of nucleoside attached to the support is determined by UVspectrophotometryby measuring the quantity of dimethoxytrityl cationliberated after a treatment with a 10% solution of trichloroacetic acidin dichloromethane. The degree of functionalization varies from 21 to 38μmol per gram of support depending on the nature of the nucleoside base.##STR31##

II.4. Automated Synthesis of the Thiooligonucleotides (Scheme 11)

The thiooloigonucleotides were synthesized in a DNA synthesizer (AppliedBiosystems model 381 A). The elongation cycle comprises four majorsteps:

Detritylation of the nucleoside or of the oligonucleotide attached tothe solid support.

Condensation of the phosphoramidite activated by tetrazole with the free5'-hydroxyl of the nucleoside or of the oligonucleotide.

Masking of the 5'-hydroxyl functional groups which have not reacted.

Oxidation of the phosphite triester functional groups intophosphotriester. ##STR32##

Each synthesis was carried out on a column containing 40 mg of solidsupport. The yield of each incorporation is 98%, evaluated by measuringthe concentration of the dimethoxytrityl cations recovered after eachcoupling by treating the support with trichloroacetic acid.

Washes are carried out between each step and the duration of anelongation cycle is 21.1 minutes for the synthesis of the homododecamer4'-thio-β-D-ribonucleotide (βrSU₁₂) (Table 1).

                  TABLE 1                                                         ______________________________________                                        Elongation cycle used for the synthesis of βrSU.sub.12.                                    Time    Solvent and                                         Number Step       (sec.)  reagents                                            ______________________________________                                        1      Drying and 24      MeCN-Argon                                                 washing                                                                2      Detritylation                                                                            80      3% TCA in CH.sub.2 Cl.sub.2                         3      Drying and 74      MeCN-Argon                                                 washing                                                                4      Coupling   915     amidite 0.1M (24 eq)-                                                         MeCN+tetrazole                                                                0.5M (10 eq)+MeCN                                   5      Drying     45      Argon                                               6      Masking    23      Methylimidazole-THF Ac.sub.2 O/-                                              lutidine/THF 118                                    7      Drying     11      Argon                                               8      Oxidation  43      0.1M I.sub.2 in THF Pyr H.sub.2 O                                             40/10/1                                             9      Washing    53      MeCN                                                ______________________________________                                    

II.5. Detachment, Deprotection and Purification of the Thiooligomer

After carrying out the steps of deprotection of the methoxyphosphatediesters with thiophenol, the detachment of the dodecamer from the solidsupport by ammonium hydroxide and the deprotection of the hydroxyls byTBAF, the thiooligomer was desalted on a G-25 DEAE Sephadex exclusioncolumn or on an A-25 Sephadex DEAE ionic column and purified byreverse-phase HPLC.

II.6 EXPERIMENTAL PART Example I Synthesis of the Homododecamer βrSU₁₂

I-1 Synthesis of the solid support:

The solid support P-1 (2.415 g) is suspended with a 3% solution (50 ml)of trichloroacetic acid in anhydrous CH₂ Cl₂. After stirring for 4 h 15min, 50 ml of a solution of triethylamine and diisopropylamine (9:1) isadded and after stirring for 5 min, the solution is filtered. The solidsupport P-2 thus obtained is washed with dichloromethane (2×50 ml) andthen with ether (50 ml) and left to dry.

The solid support P-2 is then suspended in anhydrous pyridine (14.5 ml)in the presence of succinic anhydride (0.48 g) and DMAP (0.0805 g). Thesuspension is stirred for 20 hours and then filtered. The solid supportP-3 is then washed with anhydrous pyridine (50 ml), dichloromethane(2×50 ml) and ether (2×50 ml) and then left to dry under vacuum.

Condensation of the solid support P-3 with1-[3'-O-tert-butyldimethylsilyl-2'-hydroxy-4'-thio-5'-O-dimethoxytrityl-.beta.-D-ribofuranosyl]Uracil25 b

A mixture of P-3 (2.415 g),1-[3'-O-tert-butyldimethylsilyl-2'-hydroxy-4'-thio-5'-O-dimethoxytrityl-.beta.-D-ribofuranosyl]Uracil25 b (333 mg, 1 eq.), DMAP (29.5 mmg, 0.5 eq.), triethylamine (0.146 mg,202 μl, 0.003 eq.) and DEC (920 mg, 10 eq.) in 29 ml of anhydrouspyridine is stirred for 3 days at room temperature. Pentachlorophenol(321 mg, 2.5 eq.) is then added and the mixture is kept stirring for anadditional 24 hours. The solid support is then filtered, rinsed withanhydrous pyridine (2×50 ml) and then with dichloromethane (2×50 ml) andether (2×50 ml). Immediately afterwards, the solid support P-5 istreated with 25 ml of piperidine. After stirring at room temperature for24 hours, the solid support is filtered, washed with dichloromethane(2×50 ml) and then with ether (2×50 ml) and left to dry under vacuum.The dry solid support (2,415 g) is brought into contact with a solutionof acetic anhydride (0.5M) in THF (15 ml) and with a solution ofcollidine (0.5M) in THF (15 ml). After reacting for 4 hours, the solidsupport is washed with anhydrous pyridine (2×50 ml), dichloromethane(2×50 ml), THF (2×50 ml) and ether (2×50 ml). P-6 thus obtained is leftto dry under vacuum.

I-2) Determination of the Level of Functionalization of the SolidSupport P-6

This determination is carried out by assaying the DmTr groups liberatedin an acidic medium. 38.0 mg of P-6 are weighed exactly and suspended in3.4 ml of a solution of para-toluenesulfonic acid (0.1M) inacetonitrile. The mixture is stirred for 15 min and then sonicated for 2min.

The volume is then adjusted to 10 ml with the 0.1M solution ofpara-toluenesulfonic acid in acetonitrile. 0.3 ml of this solution isremoved to which 5 ml of 0.1M para-toluenesulfonic acid solution areadded. The OD reading (0.317 unit of OD) at 500 nm allows us tocalculate the degree of functionalization of the support P-6 which is inthis case 21 μmol/gram of solid support.

I-3) Automated Synthesis of the Homododecamer βrSU₁₂

The synthesis is carried out on a column containing 39.8 mg of solidsupport functionalized at 21 μmol/g of resin, that is to say 0,835 μmol(1 eq) of 4'-thiouridine.

Each elongation cycle requires an excess of phosphoramidite synthon (20μmol, 24 eq), that is to say 240 μmol in total for the synthesis of thedocedamer. The synthesizer collecting 200 μl of a 0.1M solution ofsynthon in acetonitrile by incorporation.

The duration of an elongation cycle is 21.1 min (Table 1).

The coupling step was cosiderably increased (900 s against 45 s in thecase of a deoxynucleoside) because of the lower reactivity of theribonucleotide synthon. The assay of the liberated dimethoxytritylcations at each detritylation step made it possible to evaluate theaverage yield of incorporation of the thioribonucleotide unit at 98.7%.

I-4) Detachment, Deprotection and Purification of the ThiohomododecamerβrSU₁₂

The solid support carrying the thiohomododecamer βrSU₁₂ is treated with5 ml of a thiophenol, triethylamine, 1,2-thioxane solution (1/2/2) forhalf an hour at room temperature. The thiophenol solution is thenfiltered and the support washed with a 32% solution of ammoniumhydroxide in 95% ethanol (3/1: v/v) (3×500 μl) in order to detach thethiohomododecamer from the solid support. The solution thus obtained isevaporated, taken up in 500 μl of water and then freeze-dried. Theoligomer is then dissolved in 300 μl of a solution of TBAF (1.1M) inTHF. The reaction mixture is kept stirring for 24 hours. The reaction isthen stopped with 300 μl of an aqueous ammonium acetate solution(0.05M).

The solution is evaporated to dryness, coevaporated 3 times with waterand the residue chromatographed on a Sephadex G-25 exclusion gel column.The fractions containing the thiooligomer are combined, evaporated todryness and analyzed by HPLC. Analysis and purification of thethiooligomer βrSU₁₂ obtained.

HPLC analysis of the thiooligomer was carried out on a Beckman C-18 RP3μ×LODS Ultrasphere column under the following gradient conditions:

A: 10% acetonitrile in a 0.05M aqueous triethyl-ammonium acetatesolution

B: 15% acetonitrile in a 0.05M aqueous triethyl-ammonium acetatesolution: ##STR33##

Analysis time: 30'.

Under these conditions, it is observed that the thio-oligomer is 92.73%pure.

Purification of βrSU₁₂ is then carried out on a Nucleosylsemi-preparative column with a gradient: ##STR34## βrSU₁₂ is obtainedunder these conditions with a 94.42% purity which is sufficient for thesubsequent studies.

Example II Synthesis of the Dodecamer β (SrT)₁ dT₁₁ II-1) Synthesis ofthe Solid Support

The functionalized solid support at 1 μmol per 35 mg of2'-deoxythymidine resin as well as the synthons1-[5'-O-DmTr-3'-O-N,N-diisopropylaminocyanoethyphosphine,2'-deoxy-β-D-ribofuranosylthymine are marketed by Applied Biosystems.

II-2) Automated Synthesis of β(SrT)₁ dT₁₁

The synthesis is carried out on a column containing 35 mg of solidsupport, that is to say 1 μmol of 2'-deoxythymidine. Each elongationcycle requires an excess of phosphoramidite synthons (20 μmol, 20 eq)that is to say 220 μmol of 2'-deoxythymidine synthons and 20 μmol ofsynthon 4'-thiothymidine 5. The synthesizer collecting 200 μmol byincorporation of a 0.1M solution of each synthon in acetonitrile. Theduration of a cycle for incorporation of a deoxythymidine unit is 5.5min. Whereas it is 20.1 min for a cycle for incorporation of the synthon4'-thiothymidine.

The increase in the duration of the cycle for incorporation of thechimeric synthon is due to the coupling step of 909' against 36' for thesynthon 2'-deoxythymidine.

The assay of the liberated dimethoxytrityl cations at each detritylationstep made it possible to evaluate the average yield of incorporation ofthe 2'-deoxythymidine unit at 98.5% and 93.5% for the synthon4'-thiothymidine.

II-3) Detachment, Deprotection and Purification of β(SrT)₁ dT₁₁ (SEQ IDNO: 3)

The solid support carrying the β (SrT)₁ dT₁₁ (SEQ ID NO: 3) oligomer istreated with a solution of ammonium hydroxide (32%) in 95% ethanol (3/1,v/v) (3×500 μl) for 3 cycles of 20 min each so as to detach the oligomerfrom its support, then the dodecamer is incubated for 2 h in a dry ovenin the same mixture so as to deprotect the cyano-ethyl and methoxyfunctional groups.

The oligomer is then evaporated under vacuum, taken up in 500 μl ofwater and freeze-dried.

The deprotection of the TBDMS groups is carried out according to theprocedure developed in Example I. The residue obtained ischromatographed on a DEAE Sephadex A-25 gel column. The fractionscontaining the oligomer are combined, evaporated to dryness and analyzedby HPLC.

Analysis and purification of the β(SrT)₁ dT₁₁ (SEQ ID NO: 3) by HPLC.HPLC analysis of the oligomer was carried out on a Beckman C 18 RP 3 μUltrasphere column under the following gradient conditions:

A: 6% acetonitrile in a 0.05M triethylammonium acetate buffer.

B: 20% acetonitrile in a 0.05M triethylammonium acetate buffer ##STR35##Analysis time: 25 min.

Under these conditions, the oligomer is 49.3% pure. A purification ofβ(SrT)₁ dT₁₁ (SEQ ID NO: 3) is then carried out by semi-preparative HPLCwith a nucleosyl column under the following gradient conditions:##STR36## The 12-mer then has a retention time of 12.99 min and a puritygreater than 99%.

II.4.: Synthesis of the Dodecamer βdT₅ (SrT) dT₆ (SEQ ID NO: 3) II-4.1)Synthesis of the Solid Support

The solid support defined in Example II was used.

II-4.2) Automated Synthesis of βdT₅ (SrT) dT₆ (SEQ ID NO: 3)

The same synthesis conditions developed for the synthesis of β(SrT)₁dT₁₁ (SEQ ID NO: 3) were used.

The assay of the liberated dimethoxytrityl cations at each detritylationstep made it possible to evaluate the average yield of incorporation ofthe 2'-deoxythymidine unit at 99.3% and at 96.1% for the synthon4'-thiothymidine.

II-4.3) Detachment and Purification of the βdT₅ (SrT) dT₆ (SEQ ID NO: 3)

The treatment of this oligomer was carried out according to theprocedure developed in Example II. Analysis and the HPLC purification,carried out under the same gradient conditions, reveals a retention timeof 13.18 minutes and a spectrophotometric purity at 260 mm of 100%.

III. AUTOMATED SYNTHESIS OF 4'-THIOOLIGORIBONUCLEOTIDES AND MIXEDOLIGOMERS

The synthesis of a homogenous 4'-thioribo oligomer 1 directed on theacceptor splicing sequence of the HIV tat gene is described. It is shownthat it is then possible to obtain mixed oligomers comprising a limitedsequence of DNA having phosphodiester (sequence 4) or phosphorothioate(sequence 5) bonds.

Such mixed sequences 5'-(4'-S-RNA/DNA/4'-S-RNA)-3' 4 and 5 can be usedin an antisense approach and are capable of being highly selectiveinsofar as the "window" of the DNA structure will be the only substratefor the RNAse H after pairing of the antisense with a complementarytarget. It should be noted that the DNA "window" (phosphodiester orphosphorothioate) may be of variable length--in the sequences 4 and 5,there are six deoxynucleotides--and this "window" may be included at anyposition of the oligomer, even at the 5' and/or 3' ends.

Complementarily, this possibility opens the way to the design ofartificial ribozymes (homogeneous or otherwise) containingoligo-ribonucleotide sequences 4'-S-RNA A number of homogeneous4'-thiooligoribonucleotides (4'-S-RNA) were synthesized on a DNAsynthesizer (Applied Biosystems model 381A) using the general proceduredescribed above.

This is the case especially:

for 4'-Sr-(A-C-A-C-C-C-A-A-U-U-C-U)1SEQ ID NO: 1

for 4'-Sr-(U-U-U-U-U-U). (4'-SrU₆)2

and for 4'-Sr-(U-U-U-U-U-U-3'-P-CH₂ -CH₂ -OH), (4'-SrU₆ -3'n-prOH)3

The expression 4'-Sr means: oligoribonucleotide of β configuration wherethe oxygen of the furanose ring of the sugars is replaced with a sulfuratom. 3'n-prOH means that an n-propanol functional group is attached tothe phosphate group introduced at the 3' end of the oligoribonucleotide.

Furthermore, mixed oligomers comprising phosphodiester orphosphorothioate oligodeoxynucleotide sequences were obtained and themodifications made to the elongation cycle are described, taking thefollowing sequences as example ##STR37##

where 4'-Sr has already been defined as above, P_(x) corresponds to thenumber of phosphodiester bonds between the nucleotide units and PS_(y)to the number of phosphorothioate bonds between the nucleotide units.The thiooligoribonucleotides were synthesized on a DNA synthesizer(Applied Biosystems model 381 A).

III-1) Synthesis of the 4'-thiooligoribonucleotides4'-Sr-(A-C-A-C-C-C-A-A-U-U-C-U) (SEQ ID NO: 1) 1, 4'-SrU₆ 2, and 4'-SrU₆-3'n-prOH 3

The elongation cycle comprises four major steps:

a) Detritylation of the nucleoside or oligonucleotide attached to thesolid support in the case of 4'-SrU₆ and 4'-Sr-(A-C-A-C-C-C-A-A-U-U-C-U)(SEQ ID NO: 1), or alternatively detritylation of the n-propanol armattached to the solid support or of the oligonucleotide attached to then-propanol linkage in the case of 4'-SrU₆ -3'n-prOH.

b) Condensation of the phosphoramidite activated by tetrazole with thefree 5' hydroxyl of the nucleoside or of the oligonucleotide in the caseof 4'-SrU₆ and 4'-Sr-(A-C-A-C-C-C-A-A-U-U-C-U) (SEQ ID NO: 1), oralternatively with the free hydroxyl of the n-propanol arm or with thehydroxyl in 5' of the oligonucleotide in the case of 4'-SrU₆ -3'n-prOH.

c) Masking of the 5'-hydroxyl functional groups which have not reacted.

d) Oxidation of the phosphite triester functional groups intophosphotriester.

Each synthesis was carried out on a column containing 40 mg of solidsupport. The yield of each incorporation is 98% evaluated by measuringthe optical density of the dimethoxytrityl cations liberated after eachcoupling by treating the support with trichloroacetic acid. Washes werecarried out between each step and the duration of an elongation cycle is21.46 minutes for the synthesis of the three abovementioned oligomers.(Table 2).

                  TABLE 2                                                         ______________________________________                                        Elongation cycle used for the synthesis of 4'-Sr-                             (A-C-A-C-C-C-A-A-U-U-C-U) (SEQ ID NO:1) 1, 4'-SrU.sub.6  2,                   4'-SrU.sub.6 -3'n-prOH, 3                                                               TIME                                                                STEPS     (seconds)  SOLVENTS AND REAGENTS                                    ______________________________________                                        Washing-drying                                                                          25         CH.sub.3 CN/ARGON                                        Coupling  915        0.1M phosphoramidite                                                          nucleoside (20 eq.) in                                                        CH.sub.3 CN + 0.5M tetrazole                                                  (10 eq.) in CH.sub.3 CN.                                 Washing-drying                                                                          41         CH.sub.3 CN/ARGON                                        Oxidation 36         I.sub.2  (0.1M) in THF/Pyr H.sub.2 O                                          (40/10/1).                                               Washing-drying                                                                          59         CH.sub.3 CN/ARGON                                        Masking   23         Methylimidazole-THF 1                                                         Ac.sub.2 O/lutidine/THF:                                                      1/1 8 1                                                  Washing-drying                                                                          71         CH.sub.3 CN/ARGON                                        Detritylation                                                                           98         3% TCA in CH.sub.2 Cl.sub.2                              Washing-drying                                                                          33         CH.sub.3 CN/ARGON                                        ______________________________________                                    

III-2) Synthesis of the Mixed Oligomers 4'-SrU₃ -dT₆ -4'-SrU₃ /P₁₁ (SEQID NO: 2) 4 and 4'-SrU₃ -dT₆ -4'-SrU₃ /P₃ PS₅ P₃. III-2-1Oligonucleotide 4'-SrU₃ -dT₆ -4'-SrU₃ P₁₁ (SEQ ID NO: 2) 4.

The automated synthesis of this oligonucleotide follows the four stepsdescribed in II-1. Each synthesis was carried out on a column containing40 mg of solid support. The yield of each incorporation is 98%. Theduration of a cycle for elongation by the monomer 4'-Sr-U is 21.43minutes against 6.73 minutes for a dT monomer. This difference is due tothe increase in the coupling time for an entity 4'-Sr-U which is 915seconds against 36 seconds for a dT unit (Table 3).

                  TABLE 3                                                         ______________________________________                                        Elongation cycle used for the synthesis of                                    4'-SrU.sub.3 dT.sub.6 -4'-SrU.sub.3 /P.sub.11,  (SEQ ID NO:2) 4.                         TIME                                                               STEPS      (seconds) SOLVENTS AND REAGENTS                                    ______________________________________                                        Washing-drying                                                                           25        CH.sub.3 CN/ARGON                                        Coupling 4'-Sr-U                                                                         915       0.1M phosphoramidite                                     dT         36        nucleoside (20 eq.) in                                                        CH.sub.3 CN + 0.5M tetrazole                                                  (10 eq.) in CH.sub.3 CN.                                 Washing-drying                                                                           41        CH.sub.3 CN/ARGON                                        Oxidation  36        I.sub.2  (0.1M) in THF/Pyr/H.sub.2 O                                          (40/10/1).                                               Washing-drying                                                                           59        CH.sub.3 CN/ARGON                                        Masking    23        Methylimidazole-THF 1                                                         Ac.sub.2 O/lutidine/THF:                                                      1/1/8 1                                                  Washing-drying                                                                           71        CH.sub.3 CN/ARGON                                        Detritylation                                                                            98        3% TCA in CH.sub.2 Cl.sub.2                              Washing-drying                                                                           33        CH.sub.3 CN/ARGON                                        ______________________________________                                    

III-2-2Oligonucleotide 4'-SrU₃ -dT₆ -4'-SrU₃ P₃ PS₅ P₃, (SEQ ID NO: 2) 5

The same automated synthesis conditions were applied to this mixeddodecamer containing both phosphodiester and phosphorothioate bonds.

The synthesis was carried out on a scale of one μmol, that is to say ona column containing about 40 mg of solid support functionalized at 21μmol/g. The average yield of incorporation is 98%.

During the introduction of an internucleotide bond of thephosphorothioate type, the oxidation step defined in paragraph IV-2-1(Step 4, Table 3) was replaced by a sulfurization step of 51 secondsusing the Beaucage reagent ("derivatives of benzenesulfonecarboxythioanhydride") dissolved in acetonitrile at a concentration of0.05M.

The other steps of the elongation cycle remain unchanged.

III-3 Detachment, Deprotection and Purification of the4'-thiooligoribonucleotides (4'-S-RNA)

After having carried out the steps of deprotection of themethoxyphosphotriesters to phosphodiesters by treating with thiophenol,the 4'-thiooligoribonucleotides were separated from the solid supportsby a treatment with ammonium hydroxide. The TBDMS groups introduced intothe hydroxyls in 2' were deprotected by a solution of TBAF in THF. Thethiooligomers were then desalted on a DEAE Sephadex G-25 exclusioncolumn and then purified by reversed-phase HPLC.

III-4 EXPERIMENTAL PART Example I

Synthesis of the hexamers: 4'-SrU₆ 2 and 4'-SrU₆ -3'n-prOH, 3.

I-1 Synthesis of the Solid Supports

The solid support necessary for the synthesis of 4'-SrU₆, 2 is the sameas that used for the synthesis of βrSU₁₂. The synthesis of 4'-SrU₆-3'n-prOH 3requires the use of the universal solid support: ##STR38##From the intermediate P-3 described in Patent No. 92 01275, a mixture of1-O-DmTr-3-propanol (1 eq.), DMAP (29.5 mg, 0.5 eq.), triethylamine(0,146 mg, 0.003 eq.), DEC (920 mg, 10 eq.) and P-3 (2.415 g) is stirredin 29 ml of anhydrous pyridine for three days at room temperature.Pentachlorophenol (321 mg, 2.5 eq.) is added and the stirring of thereaction mixture is continued for an additional 24 hours. The solidsupport is then filtered, rinsed with anhydrous pyridine (2×50 ml) andthen with dichloromethane (2×50 ml) and ether (2×50 ml).

The solid support is then treated with piperidine, and then with asolution of acetic anhydride and collidine in THF according to theprocedure defined above.

I-2) Determination of the Level of Functionalization of the SolidSupport

This determination was carried out using the procedure described above.The level of functionalization of the solid support was evaluated at16.4 μmol of n-propanol per gram of resin.

I-3-) Automated Synthesis of the Hexamers 4'-SrU₆ 2 and 4'-SrU₆-3'n-prOH 3 I-3-1) Automated Synthesis of the Hexamer 4'-SrU₆ 2

The synthesis was carried out on a column containing 39.4 mg of solidsupport functionalized at 21 μmol/g of resin, that is to say 0,827 μmol(1 eq.) of 4'-thiouridine. Each elongation cycle requires an excess ofphosphoramidite synthon (16.75 μmol, 20.3 eq.) that is to say 100.5 μmolin total in a 0.1M solution in acetonitrile. The duration of theelongation cycle defined in Table 1 is 21.46 minutes.

The assay of the dimethoxytrityl cations liberated at each detritylationstep made it possible to calculate the average yield of incorporation ofa thioribonucleotide unit at 98.3%.

I-3-2) Automated Synthesis of the Hexamer 4'-SrU₆ -3'n-prOH 3

The synthesis is carried out on a column containing 36.2 mg of solidsupport functionalized at 16.4 μmol of n-propanol per gram of resin,that is to say 0.82 μmol (1 eq.) of n-propanol.

The total synthesis of 4'-SrU₆ -3'n-prOH, 3 requires 95.9 mg ofphosphoramidite synthon (114.8 μmol, 20 eq.) dissolved in 1.14 ml ofacetonitrile. The duration of an elongation cycle is 21.46 minutes. Theaverage yield per coupling is 98.3%.

I-4) Detachment, Deprotection and Purification of 4'-SrU₆ 2 and 4'-SrU₆-3'n-prOH 3

The treatment using successively thiophenol, ammonium hydroxide and asolution of TBAF common to the two hexamers is described above.

I-4-1) Analysis and Purification of the Hexamer 4'-SrU₆ 2

HPLC analysis of the thiooligomer was carried out on an SFCC NucleosylC-18 N125 2P 789 column under the following gradient conditions:

A: 10% acetonltrile in a 0.05M aqueous solution of triethylammoniumacetate

B: 15% acetonitrile in a 0.05M aqueous solution of triethylammoniumacetate ##STR39## Analysis time: 30 minutes, flow rate: 1 ml/min. Underthese conditions, the hexamer 4'-SrU₆, 2 has a spectrophotometric purityat 260 nm of 95.3%.

A preparative HPLC purification on a semi-preparative nucleosyl columnwith a flow rate of 2 ml/min and an isocratic eluent of 12% acetonitrilein a 0.05M aqueous solution of triethylamunoniumacetate enabled us toobtain 4'-SrU₆, 2 with a spectroscopic purity of 97.7%.

I-4-2) Analysis and Purification of the Hexamer 4'-SrU₆ -3'n-prOH 3

The analysis was carried out under the same column and gradientconditions as for the hexamer 4'-SrU₆.

The 4'-SrU₆ -3'n-prOH, 3 was obtained with a spectroscopic purity at 260nm of 51.6%. This purity increases to 92% after purification bypreparative HPLC carried out in a 10% to 13% gradient of acetonitrile ina 0.05M aqueous solution of triethylammonium acetate in 50 minutes,followed by a plateau at 13% acetonitrile in a 0.05M aqueous solution oftriethylammonium acetate for 10 minutes.

Example II Synthesis of the Mixed Dodecamer 4'-SrU₃ -dT₆ -4'-SrU₃ /P₁₁(SEQ ID NO: 2) 4 II-1) Synthesis of the Solid Support

The solid support used for this synthesis is identical to that requiredfor the synthesis of βrSU₁₂ (SEQ ID. NO: 5)

II-2) Automated Synthesis of 4'-SrU₃ -dT₆ -4'-SrU₃ /P₁₁ (SEQ ID NO: 2) 4

The synthesis of the dodecamer is carried out on a column containing39.3 mg of solid support, that is to say 0,819 μmol of 4'-thiouridine.Each elongation cycle requires an excess of 4'-thiouridinephosphoramidite synthons (16.75 μmol, 20.3 eq.) that is to say 100.5μmol of 4'-thiouridine synthons dissolved in 1.2 ml of acetonitrile(0,083M) and an excess of deoxythymidine phosphoramidite synthon (20eq., 16.38 μmol) that is to say 98.28 μmol of dT in a 0.1M solution inacetonitrile. The duration of a cycle for incorporation of adeoxythymidine unit is 6.73 min, whereas it is 21.43 min for a cycle forincorporation of the synthon 4'-thioribouridine.

The assay of the liberated dimethoxtytrityl cations at eachdetritylation step made it possible to evaluate the average yield ofincorporation at 98.0%.

II-3) Detachment, Deprotection and Purification of the 4'-SrU₃ -dT₆-4'-SrU₃ /P₁₁ (SEQ ID NO: 2) 4

The steps for detachment of the oligomer from the solid support and fordeprotection of the internucleotide phosphotriester bonds withthiophenol are identical to those described in Example 1.

II-3-1) Analysis and Purifaction of the 4'-SrU₃ -dT₆ -4'-SrU₃ P₁₁ (SEQID NO: 2) by HPLC

HPLC analysis of the oligomer 4 was carried out on an SFCC Nucleosyl C18N125 2P789 column with a gradient at 10% to 15% acetonitrile in a 0.05Maqueous solution of triethylammonium acetate for 20 minutes (flow rate:1 ml/min, analysis time 30 min). Under these conditions, the mixeddodecamer has a spectrophotometric purity at 260 nm of 92.5%.

A semi-preparative HPLC purification on a Nucleosyl column with a flowrate of 2 ml/min in the same gradient increases the spectrophotometricpurity of the mixed sequence 4'-SrU₃ -dT₆ -4'-SrU₃ /P₁₁ (SEQ ID NO: 2),4 to 99.5%.

EXAMPLE III Automated Synthesis of 4'-SrU₃ -dT₆ -4'-SrU₃ /P₃ PS₅ P₃ (SEQID NO: 2) 5 III-1) Synthesis of the Solid Support

The solid support used is the same as that described in Example I.

III-2) Automated Synthesis of 4'-SrU₃ -dT₆ -4'-SrU₃ /P₃ PS₅ P₃ (SEQ IDNO: 2) 5

The synthesis of the dodecamer is carried out on a column containing39.5 mg of functionalized support, that is to say 0,831 μmol (1 eq.) of4'-thiouridine. Each elongation cycle requires an excess of4'-thiouridine phosphoroamidite synthon (16.75 μmol, 20.3 eq.), that isto say 100.5 μmol of 4'-Sr-U in total and an excess of 20 eq. (16.38μmol) of deoxythymidine synthon, that is to say 98.28 μmol for theentire synthesis.

The duration of a cycle for incorporation of a deoxythymidine is 7.7minutes against 21.43 minutes for the incorporation of a 4'-thiouridine.The assay of the liberated dimethoxytrityl cations indicates an averageyield of incorporation of the synthons of 98.7%.

III-3) Detachment, Deprotection and Purification of the 4'-SrU₃ -dT₆-4'-SrU₃ /P₃ PS₅ P₃ (SEQ ID NO: 2) 5

The treatment of this oligomer is identical to that defined in ExampleI. Analysis and the HPLC purification, carried out on an SFCC NucleosylC-18 N125 2P 789 column in a gradient of 10% to 20% acetonitrile in a0.05M aqueous solution of triethylammonium acetate for 20 minutes (flowrate: 1 ml/min, analysis time 30 min) shows a spectrophotometric purityat 260 nm of 85%.

The purification by semi-preparative HPLC on a semi-preparativeNucleosyl column with a flow rate of 2 ml/min under isocratic conditionsat 19% acetonitrile in a 0.05M aqueous solution of triethylammoniumacetate increases the purity of the oligomer to 99.1%.

Example IV Synthesis of the Anti"tat" Dodecamer4'-Sr-(A-C-A-C-C-C-A-A-U-U-C-U) (SEQ ID NO: 1) 1 IV-1) Synthesis of theSolid Support

The solid support used for this automated synthesis is identical to thatdescribed in Example I.

IV-2) Automated Synthesis of the Anti"tat" Dodecamer4'-Sr-(A-C-A-C-C-C-A-A-U-U-C-U) (SEQ ID NO: 1) 1

The synthesis of this heterododecamer which is complementary to thesplicing acceptor site of the HIV tat gene is carried out on a columncontaining 38.5 mg of functionalized solid support, that is to say on0.808 μmol of 4'-thiouridine (1 eq.). Each elongation cycle uses anexcess of 25.24 eq. (20.4 μmol) of 4'-thioadenosine phosphoramiditesynthon, 13.5 eq. (10.97 μmol) of 4'-thiocytidine synthon and 30 eq.(24.25 μmol) of 4'-thiouridine synthon. The duration of an incorporationcycle is 20.71 minutes and the assay of the dimethoxytrityl cationsindicates an average yield of incorporation of the phosphoramiditesynthons of 93.6%.

IV-3) Detachment, Deprotection and Purification of4'-Sr-(A-C-A-C-C-C-A-A-U-U-C-U) (SEQ ID NO: 1) 1

The solid support carrying the dodecamer 4'-Sr-(A-C-A-C-C-C-A-A-U-U-C-U)(SEQ ID NO: 1), 1 is treated with 5 ml of athiophenol/triethylamine/dioxane solution (1/2/1) for half an hour atroom temperature. The thiophenol solution is then filtered and the solidsupport is washed with 3×3 ml of methanol, and then with a 32% ammoniumhydroxide solution in 95% ethanol

(3/1: v/v) (3×500 μl) so as to detach the oligomer from its support. Thesolution obtained is incubated in a dry oven thermostated at 55° C. for5 hours so as to deprotect the groups for protecting the heterocyclicbases. It is then evaporated, taken up in 500 μl of water andfreeze-dried. The freeze-dried oligomer is then dissolved in 300 μl of asolution of TBAF (1.1M) in THF. The reaction mixture is left at roomtemperature for 24 hours. The reaction is then stopped with 300 μl of a0.05M aqueous solution of ammonium acetate. The solution is evaporatedto dryness, coevaporated with 3 portions of 500 μl of water and theresidue is desalted on a DEAE Sephadex G-25 exclusion gel column. Thefractions containing the 4'-thiooligomer are combined, evaporated todryness, redissolved in 1.2 ml of water and filtered on a milliporefilter before being analyzed by HPLC.

IV-4) Analysis and Purification of 4'-Sr-(A-C-A-C-C-C-A-A-U-U-C-U) (SEQID NO: 1), 1

The analytical HPLC analysis of the dodecamer 1 was carried out on anSFCC Nucleosyl C18 N125 2P789 column in a gradient of 10% to 15%acetonitrile in a 0.05M aqueous solution of triethylammonium acetatewith a flow rate of 1 ml/min and an analysis time of 30 minutes. Thechromatogram indicates a spectrophotometric purity at 260 nm of 44.9%. Apurification by semi-preparative HPLC on a semi-preparative NucleosylC18 column using the same gradient with a flow rate of 2 ml/min and ananalysis time of 30 minutes brings the spectroscopic purity of the4'-Sr-(A-C-A-C-C-C-A-A-U-U-C-U) (SEQ ID NO: 1), 1 to 100%.

IV. HYBRIDIZATION PROPERTY OF THE β-4'-THIOOLIGONUCLEOTIDES

UV spectrophotometry makes it possible to study the nucleic acids and todetect the formation of duplex, the melting temperature being one of thecharacteristics of a duplex. The stacking and pairing of the bases ofthe nucleic acids are accompanied by a decrease in the UV absorbtion(hypochromicity). Thus, by UV spectrophotometry, it is possible tomonitor the formation or the dissociation of a duplex (W. SANGER,Principles of Nucleic Acid Structure, Springer-Verlag, New York, 1984,pp. 141-149). When the temperature of a solution containing a doublehelix (DNA or RNA) is slowly increased, the UV absorbtion increasessubstantially during the entire dissociation. The point of inflection ofthe curve, of sygmoidal shape, of the variation of the absorbance as asfunction of temperature is called melting temperature or Tm andcorresponds to the co-existence of separate strands and paired strandsin a 50/50 ratio.

Analysis of the melting curves for theβ-4'-thiooligoribonucleotide/β-oligodeoxyribonucleotide andβ-4'-thiooligoribonucleotide/β-oligoribonucleotide duplexes will make itpossible to define the thermal stability of such duplexes by themeasurement of the melting temperature. These hybridization experimentsare carried out preserving a 1/1 stoichiometry between the twocomplementary strands, in the presence of a 1M NaCl, 10 mM sodiumcacodylate buffer. A high NaCl concentration indeed favors the pairingsby solvating the negative charges around the phosphorus atoms of the twooligonucleotides. We therefore carried out these hybridizationexperiments in a 0.1M NaCl, 10 mM sodium cacodylate buffer.

IV.1 RESULTS AND DISCUSSION Examples I and II

Experiments for melting the βSrU₁₂ (SEQ ID NO: 5)/polyrA and βrU₁₂ (SEQID NO: 5)/polyrA duplexes.

These experiments make it possible to demonstrate the influence of thesulfur atom in position 4' of the modified dodecamer on the thermalstability of the duplex by comparing the Tm values obtained with thosefor the natural duplex.

Table 4 summarizes the melting temperatures obtained in the twoexperiments.

                  TABLE 4                                                         ______________________________________                                        Melting temperatures for the dodecamer duplexes                               Concentration 3 μM of each oligomer.                                       Concen- βSrU.sub.12 (SEQ ID NO:5)                                                                 βrU.sub.12 (SEQ ID NO:5)                        tration polyrA °C.                                                                              polyrA °C.                                    of NaCl hypochromicity % hypochromicity %                                     ______________________________________                                        1M      46° C. 25%                                                                              in progress                                          0.1M    33° C. 25%                                                                              in progress                                          ______________________________________                                    

Analysis of these results shows that the duplex βSrU_(12') (SEQ ID NO:5)/polyrA has a good thermal stability represented by the Tm of 46° C.obtained at an NaCl concentration of 1M.

Examples III and IV Experiments for melting of the βSrU_(12') (SEQ IDNO: 5)/βdC₂ A₁₂ C₂ (SEQ ID NO: 4) and βrU_(12') (SEQ ID NO: 5)/βdC₂ A₁₂C₂ (SEQ ID NO: 4) Duplexes

The melting temperatures obtained in both experiments are summarized inTable 5.

                  TABLE 5                                                         ______________________________________                                        Melting temperatures for the dodecamer duplexes                               Concentration 3 μM of each oligomer.                                               βSrU.sub.12 βrU.sub.12  (SEQ ID NO:5)                       Concen- βdC.sub.2 A.sub.12 C.sub.2                                                                βdC.sub.2 A.sub.12 C.sub.2                      tration (SEQ ID NO:4) °C.                                                                       (SEQ ID NO:4) °C.                             of NaCl hypochromicity % hypochromicity %                                     ______________________________________                                        1M      27° C. 9.3%                                                                             in progress                                          0.1M     5° C. 5.6%                                                                             in progress                                          ______________________________________                                    

Example IV

Experiment for self-hybridization of the oligomer βSrU₁₂ (SEQ ID NO: 5)

In order to verify the previous results, it is necessary to ensure thatthe dodecamer βSrU₁₂ (SEQ ID NO: 5) does not lead to anyself-hybridization. For that, an experiment for self-hybridization ofthe dodecamer βSrU₁₂ (SEQ ID NO: 5) was carried out at two NaClconcentrations (1M and 0.1M). The hybridization curves do not have thecharacteristic sigmoidal shape but obey the equation A=k-T where A isthe absorbance at 260 nm, T the temperature and k a constant. No pointof inflection can be visualized on such a straight line. We cantherefore conclude that the dodecamer βSrU₁₂ (SEQ ID NO: 5) does notself-pair.

Comparison of the melting temperatures of the duplexes βSrU₁₂ (SEQ IDNO: 5)/βdC₂ A₁₂ C₂ (SEQ ID NO: 4) (Table 5) and βSrU₁₂ (SEQ ID NO:5)/polyrA (Table 4) shows a thermal stability of the RNA/RNA homoduplex(Tm=46° C.) which is substantially greater than that for the RNA/DNAheteroduplex (Tm=27° C.), moreover, no self-hybridization of βSrU₁₂ (SEQID NO: 5) was observed.

Example VI and VII Experiment for Melting of the βdT₅ (SrT)dT₆ (SEQ IDNO: 3)/βdC₂ A₁₂ C₂ (SEQ ID NO: 4) and βdT₁₂ (SEQ ID NO: 3)/βdC₂ A₁₂ C₂(SEQ ID NO: 4) Duplexes

This experiment makes it possible to evaluate the thermal stability ofthe duplex βdT₅ (SrT)dT₆ (SEQ ID NO: 3)/βdC₂ A₁₂ C₂ (SEQ ID NO: 4) incomparison with that of the natural duplex βdT₁₂ (SEQ ID NO: 3)/βdC₂ A₁₂C₂ (SEQ ID NO: 4) (FIG. 1). The melting temperatures of these duplexesare indicated in Table 6.

                  TABLE 6                                                         ______________________________________                                        Melting temperature of the duplexes                                           βdT.sub.5 (SrT)dT.sub.6  (SEQ ID NO:3)/βdC.sub.2 A.sub.12           C.sub.2  (SEQ ID NO:4)                                                        and βdT.sub.12  (SEQ ID NO:3)/βdC.sub.2 A.sub.12 C.sub.2  (SEQ      ID NO:4)                                                                      Concentration of each oligomer 10 μM                                                  βdT.sub.5 (SrT)dT.sub.6 /                                                (SEQ ID NO:3) βdT.sub.12 (SEQ ID NO:3)/                                  βdC.sub.2 A.sub.12 C.sub.2                                                             βdC.sub.2 A.sub.12 C.sub.2                      Concentration                                                                            (SEQ ID NO:4) °C.                                                                    (SEQ ID NO:4) °C.                             of NaCl    hypochromicity %                                                                            hypochromicity %                                     ______________________________________                                        1M         40° C. 18%                                                                           45° C. 20.5%                                  ______________________________________                                    

In view of these results, it appears that the thermal stability of themodified duplex is less than that of the natural duplex. However, thedifference of 5° C. existing between the two melting temperatures is toolow to conclude that there is a mismatch. Indeed, some authors (Y.KAWASE, S. IWAI, H. INOUE, K. MIURA and E. OKTSUKA. Nucleic AcidResearch, 1986, 14, 7727) have shown that the existence of a singlemismatch in a duplex of 13 nucleotide units results in a decrease in Tmgreater than 10° C.

It can therefore be concluded that the 4'-thionucleotide pairs well with2'-deoxyadenosine but with an affinity lower than that for2'-deoxythymidine as confirmed by the difference in Tm of 5° C. which isobtained (Table 6).

IV.2. EXPERIMENTAL PART General Method for the Hybridization ExperimentsA--Assay of the Complementary Oligomers

In order to carry out the hybridization experiment, it is necessary toknow the concentration of each oligomer in order to preserve the 1:1stoichiometry between the two complementary strands. According to theBEER-LAMBERT law A=ε1c, the absorbances of the oligomers were measuredat 80° C. in order to remove the phenomenon of stacking of the basescreating a disturbance in the hyperchromicity due to the existence oftertiary structures. The molar extinction coefficients of the dodecamersat 80° C. can therefore be known according to the relationship ε²⁶⁵(SrU₁₂)=12ε²⁶⁵ (rU)

B--Hybridization Conditions

The hybridization experiments were carried out with a UVIKON 810spectrophotometer (KONTRON), coupled to an IBM-compatible microcomputer.The temperature is controlled by a HUBER PD 415 temperature programmerconnected to a thermostated bath (HUMER MINISTAT). The UV cuvettes aremade from quartz of 1 cm in length and a continous circulation ofnitrogen is used for temperatures of less than 20° C. Before theexperiment, 3 μM (Examples 1 to V) or 10 μM (Examples VI and VII) ofcomplementary oligomers are brought into contact in a sufficientquantity for 1000 μl of 1M NaCl and 10 mM sodium cacodylate buffer,adjusted to pH 7 (Examples I to VII). This mixture is heated at 80° C.for 1 hour, and then cooled to -20° C. according to a temperaturegradient of 0.5° C. per minute. The absorbance and temperature valuesare collected every 30 seconds.

The same experiment is carried out using a sufficient quantity for 1000μl of 0.1M NaCl, 10 mM sodium cacodylate hybridization buffer adjustedto pH 7 in order to evaluate the thermal stability of the duplex in thisbuffer which favors the pairings less.

EXAMPLE I Thermal Stability of the Duplex βSrU₁₂ (SEQ ID NO: 5)/polyrAI-1 Assay of the Oligomers

Concentration of βSrU₁₂ (SEQ ID NO: 5)=0.351 mM

Concentration of polyrA=4.61 mM

I-2 Hybridization Conditions

3 μM (8.55 μl) of βSrU₁₂ (SEQ ID NO: 5) and 3 μM (7.79 μl) of polyrA arebrought into contact with 983.6 μl of hybridization buffers (1M NaCl, 10mM sodium cacodylate or 0.1M NaCl, 10 mM sodium cacodylate)

EXAMPLE II Thermal Stability of the Duplex βrU₁₂ (SEQ ID NO: 5)/polyrAin Progress EXAMPLE III Thermal Stability of the Duplex βSrU₁₂ (SEQ IDNO: 5)/βdC₂ A₁₂ C₂ (SEQ ID NO: 4) III-1 Assay of the Oligomers

Concentration of βSrU₁₂ (SEQ ID NO: 5)=0.351 mM

Concentration of βdC₂ A₁₂ C₂ (SEQ ID NO: 4)=0.369 mM

III-2 Hybridization Conditions

3 μM (8.55 μl) of βSrU₁₂ (SEQ ID NO: 5) and 3 μM (8.11 μl) of βdC₂ A₁₂C₂ (SEQ ID NO: 4) are brought into contact with 983.3 μl ofhybridization buffers (1M NaCl, 10 mM sodium cacodylate or 0.1M NaCl, 10mM sodium cacodylate).

EXAMPLE IV Thermostability of the Duplex βrU₁₂ (SEQ ID NO: 5)/βdC₂ A₁₂C₂ (SEQ ID NO: 4) (in progress) IV-1 Assay of the Oligomers

Concentration of βrU₁₂ (SEQ ID NO: 5)=

Concentration of βdC₂ A₁₂ C₂ (SEQ ID NO: 4)=0.369 mM

III-2 Hybridization Conditions

3 μM (μl) of βrU₁₂ (SEQ ID NO: 5) and 3 μM (8.11 μl) of βdC₂ A₁₂ C₂ (SEQID NO: 4) are brought into contact with μl of the hybridization buffers(1M NaCl, 10 mM sodium cacodylate or 0.1M NaCl, 10 mM sodiumcacodylate).

EXAMPLE V Experiments for Self-hybridization of the Oligomer βSrU₁₂ (SEQID NO: 5) V-1 Assay of the Oligomers

Concentration of βrU₁₂ (SEQ ID NO: 5)=0.351 mM

V-2 Hybridization Conditions

6 μM (17.10 μl) of βSrU₁₂ (SEQ ID NO: 5) are brought into contact with986.9 μl of hybridization buffers (1M NaCl, 10 mM sodium cacodylate or0.1M NaCl, 10 mM sodium cacodylate).

EXAMPLE VI Thermal Stability of the Duplex

βdT₅ (SrT)dT₆ (SEQ ID NO: 3)/βdC₂ A₁₂ C₂ (SEQ ID NO: 4)

VI-1 Assay of the Oligomers

Concentration of βdT₅ (SrT)dT₆ (SEQ ID NO: 3)=1.33 mM

Concentration of βdC₂ A₁₂ C₂ (SEQ ID NO: 4)=0.369 mM

VI -2 Hybridization Conditions

10 μM (7.49 μl) of βdT₅ (SrT)dT₆ (SEQ ID NO: 3) and 10 μm (22.5 μl) ofβdC₂ A₁₂ C₂ (SEQ ID NO: 4) are brought into contact with 970 μl of 1MNaCl, 10 mM sodium cacodylate hybridization buffer.

EXAMPLE VII Thermostability of the Duplex βdT₁₂ (SEQ ID NO: 3)/βdC₂ A₁₂C₂ (SEQ ID NO: 4) VII-1 Assay of the Oligomers

Concentration of βdT₁₂ (SEQ ID NO: 3)=1.196 mM

Concentration of βdC₂ A₁₂ C₂ (SEQ ID NO: 4)=0.369 mM

VII-2 Hybridization Conditions

10 μM (8.36 μl ) of βdT₁₂ (SEQ ID NO: 3) and 10 μM (22.5 μl) of βdC₂ A₁₂C₂ (SEQ ID NO: 4) are brought into contact with 969.1 μl of 1M NaCl, 10mM sodium cacodylate hybridization buffer.

V. PROPERTIES OF HYBRIDIZATION OF THE 4'-S-RNA AND MIXED OLIGOMERS

The hybridization experiments were carried out preserving the 1/1stoichiometry between the two complementary strands in the presence of a1M NaCl, 10 mM sodium cacodylate buffer. A high NaCl concentrationindeed favors the pairings by solvating the negative charges around thephosphorus atoms of the two oligonucleotides. We carried out thesehybridization experiments in another buffer 0.1M NaCl, 10 mM sodiumcacodylate.

V-1-RESULTS AND DISCUSSION

Experiments for melting the duplexes:

    ______________________________________                                        [4'-SrU.sub.3 -dT.sub.6 -4'-SrU.sub.3 /P.sub.11  (SEQ ID NO:2)]/polyrA,       [4/polyrA]                                                                    [4'-SrU.sub.3 -dT.sub.6 -4'-SrU.sub.3 /P.sub.11  (SEQ ID NO:2)]/d(C.sub.2     A.sub.12 C.sub.2),                                                            [4/d(C.sub.2 A.sub.12 C.sub.2  (SEQ ID NO:4))]                                [4'-SrU.sub.3 -dT.sub.6 -4'-SrU.sub.3 /P.sub.3 PS.sub.5 P.sub.3  (SEQ ID      NO:2)]/polyrA,                                                                [5/polyrA]                                                                    [4'-SrU.sub.3 -dT.sub.6 -4'-SrU.sub.3 /P.sub.3 PS.sub.5 P.sub.3  (SEQ ID      NO:2)]/d(C.sub.2 A.sub.12 C.sub.2),                                           [5/d(C.sub.2 A.sub.12 C.sub.2  (SEQ ID NO:4))]                                ______________________________________                                    

are carried out at two NaCl concentrations and make it possible toevaluate the influence of the phosphorothioate bonds on the stability ofhybridization compared to the oligonucleotides with normalphosphodiester bonds (Table 7).

                  TABLE 7                                                         ______________________________________                                        Results of the melting experiments.                                           NaCl concentration (M)                                                                      Duplex             Tm °C.                                ______________________________________                                        1             4/poly rA          45                                           1             5/poly rA          36                                           1             4/d(C.sub.2 A.sub.12 C.sub.2  (SEQ ID NO:4))                                                     31                                           1             5/d(C.sub.2 A.sub.12 C.sub.2  (SEQ ID NO:4))                                                     19                                           0.1           4/poly rA          32                                           0.1           5/poly rA          24                                           0.1           4/d(C.sub.2 A.sub.12 C.sub.2 (SEQ ID NO:4))                                                      10                                           0.1           5/d(C.sub.2 A.sub.12 C.sub.2  (SEQ ID NO:4))                                                      3                                           ______________________________________                                    

The mixed dodecamer 5 having both internucleotide bonds of thephosphodiester and phosphorothioate type (P₃ PS₅ P₃) induces a decreasein the Tm value by about 2° C. per phosphorothioate linkage. On theother hand, the oligomer 4 has Tm values which are equivalent to thoseobtained for the dodecamer βrSU₁₂ (SEQ ID NO: 5) with the twocomplementary sequences poly rA and d(C₂ A₁₂ C₂)(SEQ ID NO: 4). Theseresults (Table 7) indicate that the oligomers of mixed sequence 4 and 5have a good thermal stability with complementary RNA sequences.

EXPERIMENTAL PART General Method for the Hybridization ExperimentsA--Assay of the Complementary Oligomers

In order to carry out the hybridization experiments, it is necessary toknow the concentration of each oligomer in order to preserve the 1:1stoichiometry between the two complementary strands. According to theBEER-LAMBERT law A=ε1c, the absorbances of the oligomers were measuredat 80° C. in order to remove the phenomenon of stacking of the basescreating a disturbance in the hyperchromicity due to the existence oftertiary structures. The molar extinction coefficients of the dodecamersat 80° C. can therefore be known according to a relationship similar toε²⁶⁵ (SrU₁₂)˜12ε²⁶⁵ (SrU)

B--Hybridization Conditions

The hybridization experiments were carried out with a UVIKON 810spectrophotometer (KONTRON), coupled to an IBM-compatible microcomputer.The temperature is controlled by a HUBER PD 415 temperature programmerconnected to a thermostated bath (HUMER MINISTAT). The UV cuvettes aremade from quartz of 1 cm in length and a continous circulation ofnitrogen is used for temperatures of less than 20° C. Before theexperiment, 3 μM (Examples 1 to IV) of complementary oligomers arebrought into contact in a sufficient quantity for 1000 μl of 1M NaCl and10 mM sodium cacodylate buffer, adjusted to pH 7 (Examples I to VII).This mixture is heated at 80° C. for 1 hour, and then cooled to -20° C.according to a temperature gradient of 0.5° C. per minute. Theabsorbance and temperature values are collected every 30 seconds. Thesame experiment is carried out using a sufficient quantity for 1000 μlof 0.1M NaCl, 10 mM sodium cacodylate hybridization buffer adjusted topH 7 in order to evaluate the thermal stability of the duplex in thisbuffer which favors the pairings less.

EXAMPLE I Thermal Stability of the Duplex [4'-SrU₃ -dT₆ -4'-SrU₃ /P₁₁(SEQ ID NO: 2)]/polyrA, 4/polyrA I-I Assay of the Oligomers

Concentration of 4=0.821 mM

Concentration of polyrA=4.61 mM.

I-2 Hybridization Conditions

3 μM (3.65 μl) of 4 and 3 μM (7.79 μl) of polyrA are brought intocontact with 988.5 μl of the hybridization buffers (1M NaCl, 10 mMsodium cacodylate or 0.1M NaCl, 10 mM sodium cacodylate).

EXAMPLE II Thermal Stability of Duplex 4'-SrU₃ -dT₆ -4'-SrU₃ /P₁₁ (SEQID NO: 2)/dC₂ A₁₂ C₂, 4/dC₂ A₁₂ C₂ (SEQ ID NO: 4) II-I Assay of theOligomers

Concentration of 4=0.821 mM

Concentration of dC₂ A₁₂ C₂ (SEQ ID NO: 4)=0.369 mM

II-2 Hybridization Conditions

3 μM (3.65 μl) of 4 and 3 μM (8.11 μl) of dC₂ A₁₂ C₂ (SEQ ID NO: 4) arebrought into contact with 988.2 μl of the hybridization buffera (1MNaCl, 10 mM sodium cacodylate or 0.1M NaCl, 10 mM sodium cacodylate).

EXAMPLE III Thermal Stability of the Duplex [4'-SrU₃ -dT₆ -4'-SrU₃ /P₃PS₅ P₃ (SEQ ID NO: 2)]/polyrA, 5/polyrA III-1 Assay of the Oligomers

Concentration of 5=0.40 mM.

Concentration of polyrA=4.61 mM.

III -2 Hybridization Conditions

3 μM (7.5 μl) of 5 and 3 μM (7.79 μl) of polyrA are brought into contactwith 984.7 μl of the hybridization buffers (1M NaCl, 10 mM sodiumcacodylate or 0.1M NaCl, 10 mM sodium cacodylate).

EXAMPLE IV Thermal Stability of the Duplex

[4'-SrU₃ -dT₆ -4'-SrU₃ /P₃ PS₅ P₃ (SEQ ID NO: 2)]/dC₂ A₁₂ C₂ (SEQ ID NO:4), 5/polyrA

IV-I Assay of the Oligomers

Concentration of 5=0.40 mM

Concentration of dC₂ A₁₂ C₂ (SEQ ID NO: 4)=0.369 mM

IV-2 Hybridization Conditions

3 μM (7.5 μl ) of 5 and 3 μM (8.11 μl) of dC₂ A₁₂ C₂ (SEQ ID NO: 4) arebrought into contact with 984.4 μl of the hybridization buffers (1MNaCl, 10 mM sodium cacodylate or 0.1M NaCl, 10 mM sodium cacodylate).

VI. ENZYMATIC STUDIES

The recognition of nucleic acids by enzymes is for the most part of astructural nature. The replacement of the natural heteroatom of thesugar of an oligonucleotide confers on the latter a high resistance todegradation by nucleases.

The study of the hydrolysis of the β4'-thiooligoribonucleotides by thesenucleases makes it possible to confirm that the replacement of theintracyclic oxygen by a sulfur atom indeed confers a higher enzymaticstability.

EXAMPLE I

Study of the enzymatic degradation of the dodecamer β(SrT)dT₁₁ (SEQ IDNO: 3) by calf spleen phosphodiesterase. Calf spleen phosphodiesteraseis an exonuclease which degrades the oligomers from their free 5' ends,thus liberating nucleotides 3' phosphate.

This study will allow us to evaluate the resistance to enzymaticdegradation induced by the presence in the 5' position of a4'-thionucleotide unit in the oligomer β(SrT)dT₁₁ (SEQ ID NO: 3)compared to the enzymatic degradation of the oxygenated natural homologβdT₁₂ (SEQ ID NO: 3). The comparative stability of the two dodecamersβdT₁₂ (SEQ ID NO: 3) and β(SrT)dT₁₁ (SEQ ID NO: 3) towards hydrolysis bycalf spleen phosphodiesterase is presented in Table 8.

The difference in behavior between the two dodecamers is substantial;indeed, only 23% of the oligomer β(SrT)dT₁₁ (SEQ ID NO: 3) washydrolyzed in 25 minutes whereas in the same period of time, the 12-merβdT₁₂ (SEQ ID NO: 3) was 93% degraded.

                  TABLE 8                                                         ______________________________________                                        Kinetics of degradation of βdT.sub.12  (SEQ ID NO:3) and                 β(SrT)dT.sub.11                                                          (SEQ ID NO:3) by calf spleen phosphodiesterase                                Dodecamer  Degradation in 0 min                                                                         Degradation in 25 min                               ______________________________________                                        βdT.sub.12                                                                          0%             93%                                                 (SEQ ID NO:3)                                                                 β(SrT)dT.sub.11                                                                     0%             23%                                                 (SEQ ID NO:3)                                                                 ______________________________________                                    

The variation over time of the enzymatic degradation of the twododecamers was monitored by HPLC. We were able to plot the curve forenzymatic digestion of β(SrT)dT₁₁ (SEQ ID NO: 3) as a function of time:A=A₀ e^(-kt) (FIG. 2, A) where:

A₀ is the initial surface area of the 12-mer

A is the surface area of the 12-met at the instant t

t is the time

k is the first order rate constant for the degradation

This curve (FIG. 2) allows us to calculate, using the EUREKA curvilinearregression software, k=0.016 min⁻¹ and the half-life period for thedodecamer t_(1/2) =1n 2/k=43 min.

Likewise, it was possible to plot the curve for enzymatic digestion ofβdT₁₂ (SEQ ID NO: 3) as a function of time, A=A₀ e^(-kt) (FIG. 2). It isobserved that the kinetics of degradation of βdT₁₂ (SEQ ID NO: 3) ismuch more rapid than that for β(SrT)dT₁₁ (SEQ ID NO:3); indeed, thehalf-life period is established at 1 minute for the natural dodecamer.

The kinetics of enzymatic degradation of the modified oligomerβ(SrT)dT₁₁ (SEQ ID NO: 3) by the 5' end has a half-life periodsubstantially greater than that for the degradation of βSrU₁₂. Theseresults show that the introduction of a thioribonucleotide unit at the5' end of an oligomer stabilizes the latter against calf spleenphosphodiesterase.

An oligonucleotide solution (20 μl, 3 absorbance units at 260 nm) isdiluted in 0.125M ammmonium acetate buffer (pH 7.0), 2.5 mM EDTA and0.065% Tween 80 (80 μL). A commercial solution of calf spleenphosphodisesterase (2 μl, final concentration 0.008 unit/ml) is addedand the mixture is incubated at 37° C. At determined times, aliquotfractions are collected (5 μl), heated at 100° C. for one minute andanalyzed by HPLC.

Conditions for HPLC Analysis

The analyses were carried out on an PVDI (PolyvinylimidazoleSFCC-Shandon) column protected by a precolumn and a prefilter. Theelution is carried out based on a linear gradient over 20 minutes from0.1M to 0.4M KCl in 20 mM KH₂ PO₄ at pH 6 containing 20% acetonitrile.The flow rate was fixed at 1 ml/min and the UV detection at 260 nm.

EXAMPLE II

Study of the substrate-type activities of hexamers 4'-SrU₆, 4'-SrU₆-3'n-prOH compared with the substrate characteristics of the hexamersβrU₆ and αrU₆ in relation to various purified nucleases.

Four typical nucleases were used:

Endonuclease S1, specific for the single DNA strand hydrolyzes thephosphodiester bonds in a random manner.

Calf spleen phosphodiesterase (CSP) is a 5'-exonuclease which degradesthe oligonucleotides from their 5' end and liberates nucleotides3'-phosphates.

Snake venom phosphodiesterase (VSP) is a 3'-exonuclease-3' whichliberates nucleotides 5'-phosphate after degrading the oligomer by its3' end.

Ribobuclease A degrades an oligoribonucleotide after the pyrimidineresidues.

These four enzymes were obtained from Boehringer Mannheim.

Generally, one optical density unit at 260 nm of each hexamer is broughtinto contact with a certain concentration of the enzyme considered andis then diluted in a sufficient quantity for 1000 μl of enzymaticdigestion buffer.

Various digestion buffers were used depending on the nature of theenzyme (Table 9).

                  TABLE 9                                                         ______________________________________                                        Concentrations of enzyme and digestion buffers used                                   Final concentration                                                           of enzyme (enzymatic                                                                           Enzymatic                                            Enzyme  activity units/ml)                                                                             digestion buffer                                     ______________________________________                                        CSP     13 × 10.sup.-3                                                                           EDTA 0.025 mM                                                                 pH 7                                                                          AcONH.sub.4.sup.+  0.125 mM                          VSP      6 × 10.sup.-4                                                                           Tris, HCl 0.1N                                                                pH = 9.4                                                                      MgCl.sub.2  0.01M                                    S1      20               AcONa 0.05N                                                                   NaCl 0.3M pH                                                                  4.7                                                                           AcZn 0.1M                                            RNAse A  2 × 10.sup.-2                                                                           NaCl 300 mM                                                                   Tris, HCl 10 mM                                                               pH 7.4                                                                        EDTA 5 mM                                            ______________________________________                                    

The stock sample is divided into 10 fractions of 100 μl before beingfrozen at -18° C.

One aliquot fraction of 100 μl is collected and incubated in a dry oventhermostated at 37° C. for a time t and is then analyzed by analyticalHPLC.

The study of the surface area of the signal corresponding to the hexamermakes it possible to measure a kinetics of degradation whose half-lifeperiod is calculated (Table 10).

                  TABLE 10                                                        ______________________________________                                        Enzymatic degradations                                                                    Half-life period (min)                                            Nucleases   αrU.sub.6                                                                      βrU.sub.6                                                                        βSrU.sub.6                                                                     βSrU.sub.6 3'(CH.sub.2).sub.3           ______________________________________                                                                         OH                                           5'-exonuclease                                                                            *      17      3900  --                                           calf spleen                                                                   phospho-                                                                      diesterase                                                                    3'-exonuclease                                                                            110    1        76   250                                          snake venom                                                                   phosphodi-                                                                    esterase                                                                      Endonuclease S1                                                                           *      120     930   --                                           Ribonuclsaoe A                                                                            *      <1      670   --                                           ______________________________________                                         *No degradation observed after 4 days of incubation                      

The hexamer 4'-SrU₆, 2 exhibits a high enzymatic resistance comparedwith that of βrU₆ in relation to the four nucleases (CSP, S₁, RNase-Aand VSP).

The resistance to the 3'-exonuclease is greatly increased by introducinga propanol phosphodiester bond at the 3' end of 4'-SrU₆ 3'n-prOH, 3.

CONCLUSION

The studies showed that:

a) The homogeneous or mixed 4'-thiooligoribonucleotides bind with a goodthermodynamic stability to a complementary RNA.

b) The substitution of the cyclic oxygen by a sulfur atom induces a highresistance to enzymatic degradation (cf. the comparison of the half-lifeperiod of β-rU₆ and β-S-rU₆).

All these data imply that the 4'-thiooligoribonucleotides can beconsidered as antisense agents in the form of homogeneous sequences orincluded in mixed oligomers. Furthermore, the presence of the hydroxylfunctional group in 2' makes it possible to ensivage their use asartificial ribozymes, whether in the form of homogeneous or mixedsequences associated with oligonucleotides of various types, DNA or RNA,modified or otherwise at the level of the phosphate concatenation.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ACACCCAAUUCU12                                                                (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       UUUTTTTTTUUU12                                                                (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TTTTTTTTTTTT12                                                                (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CCAAAAAAAAAAAACC16                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       UUUUUUUUUUUU12                                                                __________________________________________________________________________

What is claimed is:
 1. An oligomeric compound comprising a firstoligothionucleotide, wherein said oligothionucleotide is anoligo-4'-thio-2'-deoxyribonucleotide which comprises4'-thio-2'-deoxyribonucleotides linked by internucleotide linkages, oran oligo-4'-thioribonucleotide which comprises 4'-thioribonucleotideslinked by internucleotide linkages.
 2. The oligomeric compound of claim1, having the formula: ##STR40## wherein: the B radicals are,independently, nucleic acid bases and are attached to the glycoside ringaccording to a non-natural alpha anomeric configuration;the X radicalsare, independently, an oxoanion O⁻, a thioanion S⁻, an alkyl group, analkoxy group, a thioalkyl group, an alkyl substituted by anitrogen-containing heterocycle, an alkoxy radical substituted by anitrogen-containing heterocycle, or a --Y--Z group; R and R' are,independently, a hydrogen atom, a --Y--Z group, a Y'--Z' group, an RNAtype oligonucleotide, or a DNA type oligonucleotide; Y and Y' are,independently, a straight or branched alkylene radical --alk--, or aradical selected from the group consisting of ##STR41## wherein: U is O,S, or N; E is an oxoanion O⁻, a thioanion S⁻, an alkyl group, an alkoxygroup, a thioalkyl group, an alkyl substituted by a nitrogen-containingheterocycle, or an alkoxy radical substituted by a nitrogen-containingheterocycle; J is a hydrogen atom or a hydroxyl group; Z and Z' are,independently, a hydroxyl group or an effector selected from the groupconsisting of polycyclic compounds having a planar configuration,acridine, proflavine, biotin, furocoumarin, daunomycin, anthracycline,ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid,porphyrins, 1,10-phenanthroline, phenanthridine, ellipticine,ellipticinium, dipyrido(1,2-a:3',2'-d)imidazole, diazapyrene,4-azidoacetophenone, ethylene-imine, beta-chloroethylamine, psoralen,methylpyrroporphyrin, and aromatic compounds absorbing near-ultravioletor visible radiations; n is an integer including 0; L is an oxygen atom,a sulfur atom, or an --NH-- group.
 3. The oligomeric compound of claim2, wherein Y and Y' are independently ##STR42## wherein U is O, S, or N,and n' is an integer from 1 to
 10. 4. The oligomeric compound of claim2, wherein L is oxygen, R and R' are hydrogen atoms, and B is a naturalnucleic acid base.
 5. The oligomeric compound of claim 2, wherein Z andZ' are independently acridine, furocoumarin, daunomycin,1,10-phenanthroline, phenanthridine, proflavine, porphyrins,dipyrido(1,2-a:3'-d) imidazole, ellipticine, ellipticinium, ordiazapyrene.
 6. The oligomeric compound of claim 2, wherein the nucleicacid bases are thymine, adenine, 2-aminoadenine, cytosine, guanine,uracil, 5-bromouracil, 8-azidoadenine, 7-deazaadenine, 7-deazaguanine,4-azidocytosine, or 4-azidothymine.
 7. The oligomeric compound of claim2, wherein J is OH.
 8. The oligomeric compound of claim 1 furthercomprising an RNA type oligonucleotide or a DNA type oligonucleotidelinked by an internucleotide linkage to said compound of claim 1,wherein oxygen is the heteroatom of the furanose ring of the nucleotidesof said DNA type oligonucleotide or RNA type oligonucleotide.
 9. Theoligomeric compound of claim 1 further comprising an RNA typeoligonucleotide or a DNA type oligonucleotide linked by internucleotidelinkages at one or each of its ends to said compound of claim 1, whereinoxygen is the heteroatom of the furanose ring of the nucleotides of saidDNA type oligonucleotide or RNA type oligonucleotide.
 10. The oligomericcompound of claim 2 further comprising an RNA type oligonucleotide or aDNA type oligonucleotide linked by an internucleotide linkage to saidcompound of claim 2, wherein oxygen is the heteroatom of the furanosering of the nucleotides of said DNA type oligonucleotide or RNA typeoligonucleotide.
 11. The oligomeric compound of claim 2 furthercomprising an RNA type oligonucleotide or a DNA type oligonucleotidelinked by internucleotide linkages at one or each of its ends to saidcompound of claim 2, wherein oxygen is the heteroatom of the furanosering of the nucleotides of said DNA type oligonucleotide or RNA typeoligonucleotide.
 12. The oligomeric compound of claim 8, furthercomprising a second oligothionucleotide linked to said DNA typeoligonucleotide, wherein said second oligothionucleotide comprises4'-thlo-2'-deoxyribonucleotides or 4'-thioribonucleotides linked byinternucleotide linkages.
 13. The oligomeric compound of claim 10,further comprising a second oligothionucleotide linked to said DNA typeoligonucleotide, wherein said second oligothionucleotide comprises4'-thio-2'-deoxyribonucleotides or 4'-thioribonucleotides linked byinternucleotide linkages.
 14. The oligomeric compound of claim 12,wherein said first and second oligothionucleotides areoligo-4'-thioribonucleotides.
 15. The oligomeric compound of claim 13,wherein said first and second oligothionucleotides areoligo-4'-thioribonucleotides.
 16. A process for preparing the oligomericcompound of claim 1, comprising the steps:(a) synthesizing anoligothionucleotide using 4'-thionucleotides, wherein theinternucleotide linkages of said oligothionucleotide are selected fromthe group consisting of phosphodiester, phosphotriester,phosphoramidite, and hydrogen phosphonate, and wherein said4'-thionucleotides are completely protected; and (b) removing theprotecting groups to obtain said oligomeric compound.
 17. A process forpreparing the oligomeric compound of claim 2, wherein both Z and Z' areOH, by a phosphoramidite method comprising the steps:(a) protecting the5' position of a 4'-thionucleotide or an oligo-4'-thionucleotide withdimethoxytrityl and protecting the 3'position of a 4'-thionucleotide oran oligo-4'-thionucleotide with methyl diisopropylaminophosphoramidite;(b) functionalizing a solid support having an amino group byincorporating a 4'-thionucleoside derivative having a succinyl linkagebetween the 3'-hydroxyl group of the 4'-thionucleoside derivative andthe amino group of the solid support; (c) elongating theoligothionucleotide chain in a synthesizing reactor; and (d) detachingthe oligothionucleotide from the solid support, deprotecting the 5' and3' positions, and purifying said oligomeric compound.
 18. The oligomericcompound of claim 1 further comprising a linked effector, which is anintercalating agent or a chemical or photoactivable radical, whereinsaid effector is a polycyclic compound having a planar configuration,ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid,porphyrins, 1,10-phenanthroline, 4-azidoacetophenone, ethylene-imine,beta-chloroethylamine, psoralen, acridine, proflavine, biotin,furocoumarin, daunomycin, anthracycline, phenanthridine, ellipticine,ellipticinium, methylpyrroporphyrin, dipyrido(1,2-a:3',2'-d) imidazole,diazapyrene, or an aromatic compound absorbing near-ultraviolet orvisible radiations.